Anti-TIM-3 Antibodies

ABSTRACT

Anti-TIM-3 antibodies are disclosed. Also disclosed are pharmaceutical compositions comprising such antibodies, and uses and methods using the same.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No, 15/521,514entitled “Anti-TIM-3 Antibodies” filed Apr. 24, 2017, which claimspriority to and is a 371 national phase of applicationPCT/SG2015/050414, filed Oct. 27, 2015, which claims priority to GBApplication No. 1419094.6, filed Oct. 27, 2014, all of which areincorporated herein by reference in their entirety.

SEQUENCE LISTING DATA

The Sequence Listing text document filed herewith, created Aug. 19,2019, size 38.2 kilobytes, and named “Sequence_Listing” is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to T cellimmunoglobulin mucin 3 (TIM-3).

BACKGROUND TO THE INVENTION

T-cell exhaustion is a state of T-cell dysfunction that arises duringmany chronic infections and cancer. It is defined by poor T-celleffector function, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T-cells. Exhaustion prevents optimal control of infection andtumors. (E John Wherry., Nature Immunology 12, 492-499 (2011)).

T-cell exhaustion is characterized by the stepwise and progressive lossof T-cell functions. Exhaustion is well-defined during chroniclymphocytic choriomeningitis virus infection and commonly develops underconditions of antigen-persistence, which occur following many chronicinfections including hepatitis B virus, hepatitis C virus and humanimmunodeficiency virus infections, as well as during tumor metastasis.Exhaustion is not a uniformly disabled setting as a gradation ofphenotypic and functional defects can manifest, and these cells aredistinct from prototypic effector, memory and also anergic T cells.Exhausted T cells most commonly emerge during high-grade chronicinfections, and the levels and duration of antigenic stimulation arecritical determinants of the process. (Yi et al., Immunology April 2010;129(4):474-481).

Circulating human tumor-specific CD8⁺ T cells may be cytotoxic andproduce cytokines in vivo, indicating that self- and tumor-specifichuman CD8⁺ T cells can reach functional competence after potentimmunotherapy such as vaccination with peptide, incomplete Freund'sadjuvant (IFA), and CpG or after adoptive transfer. In contrast toperipheral blood, T-cells from metastasis are functionally deficient,with abnormally low cytokine production and upregulation of theinhibitory receptors PD-1, CTLA-4, and TIM-3. Functional deficiency isreversible, since T-cells isolated from melanoma tissue can restoreIFN-γ production after short-term in vitro culture. However, it remainsto be determined whether this functional impairment involves furthermolecular pathways, possibly resembling T-cell exhaustion or anergy asdefined in animal models. (Baitsch et al., J Clin Invest. 2011;121(6):2350-2360).

Programmed cell death 1 (PD-1), also called CD279, is a type I membraneprotein encoded in humans by the PDCD1 gene. It has two ligands, PD-L1and PD-L2.

The PD-1 pathway is a key immune-inhibitory mediator of T-cellexhaustion. Blockade of this pathway can lead to T-cell activation,expansion, and enhanced effector functions. As such, PD-1 negativelyregulates T cell responses. PD-1 has been identified as a marker ofexhausted T cells in chronic disease states, and blockade of PD-1:PD-1Linteractions has been shown to partially restore T cell function.(Sakuishi et al., JEM Vol. 207, Sep. 27, 2010, pp2187-2194).

Nivolumab (BMS-936558) is an anti-PD-1 antibody that was approved forthe treatment of melanoma in Japan in July 2014. Other anti-PD-1antibodies are described in WO 2010/077634, WO 2006/121168.

T cell immunoglobulin mucin 3 (TIM-3) is an immune regulator identifiedas being upregulated on exhausted CD8⁺ T cells (Sakuishi et al., JEMVol. 207, Sep. 27, 2010, pp2187-2194). TIM-3 was originally identifiedas being selectively expressed on IFN-γ-secreting Th1 and Tc1 cells.Interaction of TIM-3 with its ligand, galectin-9, triggers cell death inTIM-3⁺ T cells. Anti-TIM-3 antibodies are described in Ngiow et al(Cancer Res. 2011 May 15; 71(10):3540-51),and in U.S. Pat. No.8,552,156.

Both TIM-3 and PD-1 can function as negative regulators of T cellresponses and combined targeting of the TIM-3 and PD-1 pathways is moreeffective in controlling tumor growth than targeting either pathwayalone. (Sakuishi et al., JEM Vol. 207, Sep. 27, 2010, pp2187-2194; andNgiow et al Cancer Res. 2011 May 15; 71(10):3540-51). TIM-3 can also beexpressed on the surface of tumor cells, particularly tumor cells ofhematopoietic origin, such for example acute myeloid leukemia cells(Kikushige et al., Cell Stem Cell 2010; 3:7(6)708-17), Therefore in someinstances TIM-3 could be a tumor-associated antigen that could betargeted by specific antibodies.

SUMMARY OF THE INVENTION

The present invention is concerned with antibodies, or antigen bindingfragments, that bind to TIM-3. Heavy and light chain polypeptides arealso disclosed. The antibodies, antigen binding fragments andpolypeptides may be provided in isolated and/or purified form and may beformulated into compositions suitable for use in research, therapy anddiagnosis.

In some embodiments the antibody, or antigen binding fragment, orpolypeptide is cytotoxic, e.g. against TIM-3 expressing cells, such asTIM-3 expressing T-cells or tumor cells. In some embodiments theantibody, or antigen binding fragment, or polypeptide is useful intreating cancer owing to its cytotoxic effect. Suitable cancers includeleukemia, such as acute myeloid leukemia.

In some embodiments the antibody, or antigen binding fragment, orpolypeptide may be effective to restore T-cell function in T-cells, e.g.CD8⁺ T-cells, exhibiting T-cell exhaustion or T-cell anergy.

In one aspect of the present invention an antibody, or antigen bindingfragment, is provided, the amino acid sequence of the antibody maycomprise the amino acid sequences i) to iii), or the amino acidsequences iv) to vi), or preferably the amino acid sequences i) to vi):

(SEQ ID NO: 10) i) LC-CDR1 : SGSSSNIGNNYVS (SEQ ID NO: 11) ii) LC-CDR2:GNNWRPS (SEQ ID NO: 41) iii) LC-CDR3: X₁X₂WDX₃X₄X₅X₆X₇GX₈(SEQ ID NO: 30) iv)  HC-CDR1 : GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS(SEQ ID NO: 31) v) HC-CDR2: EINHSGSTNYNPSLKS (SEQ ID NO: 42) vi)HC-CDR3: GYZ₁AGZ₂DZ₃or a variant thereof in which one or two or three amino acids in one ormore of the sequences (i) to (vi) are replaced with another amino acid,where X₁=E or D, X₂=A or S, X₃=Y, S or A, X₄=Y, A, D or S, X₅=V, D, Y, Lor A, X₆=A or S, X₇=A or S, X₈=Y, V or S and Z₁=V or Y, Z₂=S, F, Y or D,Z₃=A, D, S or Y.

In connection with all aspects of the present invention, in embodimentswherein HC-CDR1: GYYWS (SEQ ID NO:61), this sequence may be comprised inthe larger sequence GGSFSGYYWS (SEQ ID NO:30).

In some embodiments, LC-CDR3 is one of EAWDYYVAAGY (SEQ ID NO:12),DSWDSADASGV (SEQ ID NO:13), DSWDYDYAAGV (SEQ ID NO:14), DSWDSYLAAGV (SEQID NO:15), ESWDYDYASGV (SEQ ID NO:16), DSWDSSDSSGV (SEQ ID NO:17),EAWDSAYAAGS(SEQ ID NO:18) DSWDAALSAGV (SEQ ID NO:19) or ESWDAAAAAGY (SEQID NO:20).

In some embodiments HC-CDR3 is one of GYVAGSDA (SEQ ID NO:32), GYVAGFDD(SEQ ID NO:33), GYVAGFDS (SEQ ID NO:34), GYVAGYDY (SEQ ID NO:35),GYVAGSDA (SEQ ID NO:36), GYVAGFDS (SEQ ID NO:37), GYYAGDDY (SEQ IDNO:38), GYVAGYDY (SEQ ID NO:39), or GYVAGSDA (SEQ ID NO:40).

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 10) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 11) LC-CDR2: GNNWRPS(SEQ ID NO: 12) LC-CDR3: EAWDYYVAAGY

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 10) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 11) LC-CDR2: GNNWRPS(SEQ ID NO: 13) LC-CDR3: DSWDSADASGV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 10) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 11) LC-CDR2: GNNWRPS(SEQ ID NO: 14) LC-CDR3: DSWDYDYAAGV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 10) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 11) LC-CDR2: GNNWRPS(SEQ ID NO: 15) LC-CDR3: DSWDSYLAAGV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 10) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 11) LC-CDR2: GNNWRPS(SEQ ID NO: 16) LC-CDR3: ESWDYDYASGV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

(SEQ ID NO: 10) LC-CDR1: SGSSSNIGNNYVS (SEQ ID NO: 11) LC-CDR2: GNNWRPS(SEQ ID NO: 17) LC-CDR3: DSWDSSDSSGV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYV LC-CDR2: (SEQ ID NO: 11) GNNWRPSLC-CDR3: (SEQ ID NO: 18) EAWDSAYAAGS

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2: (SEQ ID NO: 11) GNNWRPSLC-CDR3: (SEQ ID NO: 19) DSWDAALSAGV

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one light chain variable region incorporating thefollowing CDRs:

LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2: (SEQ ID NO: 11) GNNWRPSLC-CDR3: (SEQ ID NO: 20) ESWDAAAAAGY

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 32) GYVAGSDA

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 33) GYVAGFDD

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 34) GYVAGFDS

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 35) GYVAGYDY

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable reaion incorooratina thefollowina CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 36) GYVAGSDA

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 37) GYVAGFDS

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 38) GYYAGDDY

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 39) GYVAGYDY

In some embodiments the antibody, or antigen binding fragment, maycomprise at least one heavy chain variable region incorporating thefollowing CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 40) GYVAGSDA

The antibody may comprise at least one light chain variable regionincorporating the CDRs shown in FIG. 1 or 2. The antibody may compriseat least one heavy chain variable region incorporating the CDRs shown inFIG. 1 or 3.

The antibody may comprise at least one light chain variable region(V_(L)) comprising the amino acid sequence of one of SEQ ID NOs 1, 10,11, 12 or 2, 10, 11, 13 or 3, 10, 11, 14 or 4, 10, 11, 15 or 5, 10, 11,16 or 6, 10, 11, 17 or 7, 10, 11, 18 or 8, 10, 11, 19 or 9, 10, 11, 20or one of the amino acid sequences shown in FIG. 1 or an amino acidsequence having at least 70%, more preferably one of at least 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100%, sequence identity to one of SEQ ID NOs 1, 10, 11, 12 or 2,10, 11, 13 or 3, 10, 11, 14 or 4, 10, 11, 15 or 5, 10, 11, 16 or 6, 10,11, 17 or 7, 10, 11, 18 or 8, 10, 11, 19 or 9, 10, 11, 20 orto the aminoacid sequence of the V_(L) chain amino acid sequence shown in FIG. 1.

The antibody may comprise at least one heavy chain variable region(V_(H)) comprising the amino acid sequence of one of SEQ ID NOs 21, 30or 61, 31, 32 or 22, 30 or 61, 31, 33 or 23, 30 or 61, 31, 34 or 24, 30or 61, 31, 35 or 25, 30 or 61, 31, 36 or 26, 30 or 61, 31, 37 or 27, 30or 61, 31, 38 or 28, 30 or 61, 31, 39 or 29, 30 or 61, 31, 40 or one ofthe amino acid sequences shown in FIG. 2 or an amino acid sequencehaving at least 70%, more preferably one of at least 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%, sequence identity to one of SEQ ID NOs 21, 30 or 61, 31, 32 or 22,30 or 61, 31, 33 or 23, 30 or 61, 31, 34 or 24, 30 or 61, 31, 35 or 25,30 or 61, 31, 36 or 26, 30 or 61, 31, 37 or 27, 30 or 61, 31, 38 or 28,30 or 61, 31, 39 or 29, 30 or 61, 31, 40 or to the amino acid sequenceof the V_(H) chain amino acid sequence shown in FIG. 2.

The antibody may comprise at least one light chain variable regioncomprising the amino acid sequence of one of SEQ ID NOs 1, 10, 11, 12 or2, 10, 11, 13 or 3, 10, 11, 14 or 4, 10, 11, 15 or 5, 10, 11, 16 or 6,10, 11, 17 or 7, 10, 11, 18 or 8, 10, 11, 19 or 9, 10, 11, 20 or one ofthe amino acid sequences shown in FIG. 1 (or an amino acid sequencehaving at least 70%, more preferably one of at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100%, sequence identity to one of SEQ ID NOs1, 10, 11, 12 or 2, 10, 11, 13 or 3, 10, 11, 14 or 4, 10, 11, 15 or 5,10, 11, 16 or 6, 10, 11, 17 or 7, 10, 11, 18 or 8, 10, 11, 19 or 9, 10,11, 20 or to one of the amino acid sequences of the V_(L) chain aminoacid sequence shown in FIG. 1) and at least one heavy chain variableregion comprising the amino acid sequence of one of SEQ ID NOs 21, 30 or61, 31, 32 or 22, 30 or 61, 31, 33 or 23, 30 or 61, 31, 34 or 24, 30 or61, 31, 35 or 25, 30 or 61, 31, 36 or 26, 30 or 61, 31, 37 or 27, 30 or61, 31, 38 or 28, 30 or 61, 31, 39 or 29, 30 or 61, 31, 40 or one of theamino acid sequence shown in FIG. 2 (or an amino acid sequence having atleast 70%, more preferably one of at least 75%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, sequenceidentity to one of SEQ ID NOs 21, 30 or 61, 31, 32 or 22, 30 or 61, 31,33 or 23, 30 or 61, 31, 34 or 24, 30 or 61, 31, 35 or 25, 30 or 61, 31,36 or 26, 30 or 61, 31, 37 or 27, 30 or 61, 31, 38 or 28, 30 or 61, 31,39 or 29, 30 or 61, 31, 40 or to one of the amino acid sequences of theV_(H) chain amino acid sequence shown in FIG. 2).

The antibody may optionally bind TIM-3. The antibody may optionally haveamino acid sequence components as described above. The antibody may bean IgG. In one embodiment an in vitro complex, optionally isolated,comprising an antibody, or antigen binding fragment, as describedherein, bound to TIM-3 is provided.

In one aspect of the present invention an isolated heavy chain variableregion polypeptide is provided, the heavy chain variable regionpolypeptide comprising the following CDRs:

HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61) GYYWS HC-CDR2:(SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 42) GYZ₁AGZ₂DZ₃where Z₁=V or Y, Z₂=S, F, Y or D, Z₃=A, D, S or Y.

In some embodiments HC-CDR3 is one of GYVAGSDA (SEQ ID NO:32), GYVAGFDD(SEQ ID NO:33), GYVAGFDS (SEQ ID NO:34), GYVAGYDY (SEQ ID NO:35),GYVAGSDA (SEQ ID NO:36), GYVAGFDS (SEQ ID NO:37), GYYAGDDY (SEQ IDNO:38), GYVAGYDY (SEQ ID NO:39), or GYVAGSDA (SEQ ID NO:40).

In one aspect of the present invention an antibody, or antigen bindingfragment, is provided, the antibody, or antigen binding fragment,comprising a heavy chain and a light chain variable region sequence,wherein:

-   -   the heavy chain comprises a HC-CDR1, HC-CDR2, HC-CDR3, having at        least 85% overall sequence identity to    -   HC-CDR1: GGSFSGYYWS (SEQ ID NO:30) or GYYWS (SEQ ID NO:61)    -   HC-CDR2:EINHSGSTNYNPSLKS (SEQ ID NO:31),    -   HC-CDR3: is one of GYZ,AGZ₂DZ₃ (SEQ ID NO:42), GYVAGSDA (SEQ ID        NO:32), GYVAGFDD (SEQ ID NO:33), GYVAGFDS (SEQ ID NO:34),        GYVAGYDY (SEQ ID NO:35), GYVAGSDA (SEQ ID NO:36), GYVAGFDS (SEQ        ID NO:37), GYYAGDDY (SEQ ID NO:38), GYVAGYDY (SEQ ID NO:39), or        GYVAGSDA (SEQ ID NO:40) respectively, where Z1=V or Y, Z2=S, F,        Y, or D, Z3=A, D, S or Y. and    -   the light chain comprises a LC-CDR1, LC-CDR2, LC-CDR3, having at        least 85% overall sequence identity to:

LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS, LC-CDR2: (SEQ ID NO: 11)GNNWRPS, LC-CDR3: (SEQ ID NO: 41) is one of X₁X₂WDX₃X₄X₅X₆X₇GX₈,(SEQ ID NO: 12) EAWDYYVAAGY, (SEQ ID NO: 13) DSWDSADASGV,(SEQ ID NO: 14) DSWDYDYAAGV, (SEQ ID NO: 15) DSWDSYLAAGV,(SEQ ID NO: 16) ESWDYDYASGV, (SEQ ID NO: 17) DSWDSSDSSGV,(SEQ ID NO: 18) EAWDSAYAAGS  (SEQ ID NO: 19) DSWDAALSAGV or(SEQ ID NO: 20) ESWDAAAAAGY, respectively, where X₁ = E or D, X₂ =A or S, X₃ = Y, S or A, X₄ = Y, A, D or S, X₅ = V, D, Y or L or A, X₆ =A or S, X₇ = A or S, X₈ = Y, V or S.

In some embodiments the degree of sequence identity may be one of 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In another aspect of the present invention an antibody, or antigenbinding fragment, optionally isolated, is provided comprising a heavychain and a light chain variable region sequence, wherein:

-   -   the heavy chain sequence has at least 85% sequence identity to        the heavy chain sequence of one of SEQ ID NOs: 21 to 29 (FIG.        2), and    -   the light chain sequence has at least 85% sequence identity to        the light chain sequence of one of: SEQ ID NO:1 to 9 (FIG. 1).

In some embodiments the degree of sequence identity may be one of 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In some embodiments the antibody, antigen binding fragment, orpolypeptide further comprises variable region heavy chain frameworksequences between the CDRs according to the arrangementHCFR1:HC-CDR1:HCFR2:HC-CDR2:HCFR3:HC-CDR3:HCFR4. The framework sequencesmay be derived from human consensus framework sequences.

In one aspect of the present invention an isolated light chain variableregion polypeptide, optionally in combination with a heavy chainvariable region polypeptide as described herein, is provided, the lightchain variable region polypeptide comprising the following CDRs:

LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2: (SEQ ID NO: 11) GNNWRPSLC-CDR3: (SEQ ID NO: 41) X₁X₂WDX₃X₄X₅X₆X₇GX₈where X₁=E or D, X₂=A or S, X₃=Y, S or A, X₄=Y, A, D or S, X₅=V, D, Y orL or A, X₆=A or S, X₇=A or S, X₈=Y, V or S.

In some embodiments, LC-CDR3 is one of EAWDYYVAAGY (SEQ ID NO:12),DSWDSADASGV (SEQ ID NO:13), DSWDYDYAAGV (SEQ ID NO:14), DSWDSYLAAGV (SEQID NO:15), ESWDYDYASGV (SEQ ID NO:16), DSWDSSDSSGV (SEQ ID NO:17),EAWDSAYAAGS(SEQ ID NO:18) DSWDAALSAGV (SEQ ID NO:19) or ESWDAAAAAGY (SEQID NO:20).

In some embodiments the antibody, antigen binding fragment, orpolypeptide further comprises variable region light chain frameworksequences between the CDRs according to the arrangementLCFR1:LC-CDR1:LCFR2:LC-CDR2:LCFR3:LC-CDR3:LCFR4. The framework sequencesmay be derived from human consensus framework sequences.

In some embodiments, the antibody, or antibody binding fragment, mayfurther comprise a human constant region. For example selected from oneof IgG1, IgG2, IgG3 and IgG4.

In some embodiments, the antibody, or antibody binding fragment, mayfurther comprise a murine constant region. For example, selected fromone of IgG1, IgG2A, IgG2B and IgG3.

In another aspect of the present invention, an antibody or antigenbinding fragment is provided, optionally isolated, which is capable ofbinding to TIM-3, and which is a bispecific antibody or a bispecificantigen binding fragment. In some embodiments, the bispecific antibodyor bispecific antigen binding fragment comprises an antigen bindingfragment or polypeptide capable of binding to TIM-3 as described herein,and additionally comprises an antigen binding domain which is capable ofbinding to another target protein, e.g. a target protein other thanTIM-3. In some embodiments, the target protein is a cell surfacereceptor. In some embodiments, the target protein is a cell surfacereceptor expressed on the cell surface of immune cells, e.g. T cells. Insome embodiments, the antigen binding domain capable of binding toanother target protein may be capable of binding to a T cell receptor(TCR) complex or a component thereof. In some embodiments, the antigenbinding domain may be capable of binding to CD3 or a CD3 polypeptide. Insome embodiments, the antigen binding domain may be capable of bindingto one or more of the CD3 polypeptides CD3γ, CD3δ, CD3ζ, or CD3ε. Insome embodiments the bispecific antibody is a bispecific T-cell engagerantibody. In some embodiments, the target protein may be a member of theCD28 family. In some embodiments, the member of the CD28 family isselected from PD-1, LAG3, ICOS, CTLA4, BTLA or CD28.

In another aspect of the present invention, a composition, e.g. apharmaceutical composition or medicament, is provided. The compositionmay comprise an antibody, antigen binding fragment, or polypeptide asdescribed herein and at least one pharmaceutically-acceptable carrier,excipient, adjuvant or diluent.

In another aspect of the present invention an isolated nucleic acidencoding an antibody, antigen binding fragment, or polypeptide asdescribed herein is provided. The nucleic acid may have a sequence ofone of SEQ ID NOs 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60 (FIG. 4), or a coding sequence which is degenerate asa result of the genetic code, or may have a nucleotide sequence havingat least 70% identity thereto, optionally one of 75%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%.

In one aspect of the present invention there is provided a vectorcomprising a nucleic acid described herein. In another aspect of thepresent invention, there is provided a host cell comprising the vector.For example, the host cell may be eukaryotic, or mammalian, e.g. ChineseHamster Ovary (CHO), or human or may be a prokaryotic cell, e.g. E.coli. In one aspect of the present invention a method for making anantibody, or antigen binding fragment or polypeptide as described hereinis provided, the method comprising culturing a host cell as describedherein under conditions suitable for the expression of a vector encodingthe antibody, or antigen binding fragment or polypeptide, and recoveringthe antibody, or antigen binding fragment or polypeptide.

In another aspect of the present invention an antibody, antigen bindingfragment or polypeptide is provided for use in therapy, or in a methodof medical treatment. In another aspect of the present invention anantibody, antigen binding fragment or polypeptide as described herein isprovided for use in the treatment of cancer or a T-cell dysfunctionaldisorder. In another aspect of the present invention, the use of anantibody, antigen binding fragment or polypeptide as described herein inthe manufacture of a medicament or pharmaceutical composition for use inthe treatment of cancer or a T-cell dysfunctional disorder is provided.

In another aspect a method, in vitro or in vivo, of killing a cell thatexpresses TIM-3 is provided, the method comprising administering anantibody, antigen binding fragment or polypeptide as described herein toa cell that expresses (or overexpresses) TIM-3. The cell may be a cancercell, e.g. leukemia or acute myeloid leukemia cell, white blood cell orT-cell. In some embodiments, the acute myeloid leukemia cell may be astem cell; for example, in some embodiments the acute myeloid leukemiacell may be CD34+.

In another aspect of the present invention a method of enhancing T-cellfunction comprising administering an antibody, antigen binding fragmentor polypeptide as described herein to a dysfunctional T-cell isprovided. The method may be performed in vitro or in vivo.

In another aspect of the present invention a method of treating canceror a T-cell dysfunctional disorder is provided, the method comprisingadministering an antibody, antigen binding fragment or polypeptide asdescribed herein to a patient suffering from cancer or a T-celldysfunctional disorder.

In another aspect of the present invention a method of treating aninfectious disease is provided, the method comprising administering anantibody, antigen binding fragment of polypeptide as described herein toa patient suffering from an infectious disease.

In another aspect of the present invention a method of modulating animmune response in a subject is provided, the method comprisingadministering to the subject an antibody, antigen binding fragment orpolypeptide as described herein such that the immune response in thesubject is modulated.

In another aspect of the present invention a method of inhibiting growthof tumor cells in a subject is provided, the method comprisingadministering to the subject a therapeutically effective amount of anantibody, antigen binding fragment or polypeptide as described herein.

In another aspect of the present invention a method is provided, themethod comprising contacting a sample containing, or suspected tocontain, TIM-3 with an antibody or antigen binding fragment, asdescribed herein, and detecting the formation of a complex of antibody,or antigen binding fragment, and TIM-3.

In another aspect of the present invention a method of diagnosing adisease or condition in a subject is provided, the method comprisingcontacting, in vitro, a sample from the subject with an antibody, orantigen binding fragment, as described herein, and detecting theformation of a complex of antibody, or antigen binding fragment, andTIM-3.

In a further aspect of the present invention a method of selecting orstratifying a subject for treatment with a modulator of TIM-3 signallingis provided, the method comprising contacting, in vitro, a sample fromthe subject with an antibody, or antigen binding fragment, according tothe present invention and detecting the formation of a complex ofantibody, or antigen binding fragment, and TIM-3.

An aspect of the present invention is a method of selecting a patientfor treatment with a modulator of TIM3 signalling, such as an anti-TIM3antibody or anti-TIM3 agent, the method comprising contacting, in vitro,a sample from the subject with an antibody, or antigen binding fragmentas described herein, and detecting the formation of a complex of theantibody, or antigen binding fragment, and TIM-3.

In a further aspect of the present invention the use of an antibody, orantigen binding fragment, as described herein, for the detection ofTIM-3 in vitro is provided. In another aspect of the present inventionthe use of an antibody, or antigen binding fragment, as describedherein, as an in vitro diagnostic agent is provided.

In a further aspect of the present invention a method for expanding apopulation of T cells is provided, wherein T cells are contacted invitro or ex vivo with an antibody, antigen binding fragment orpolypeptide according to the present invention.

In a further aspect of the present invention a method of treatment of asubject having a T-cell dysfunctional disorder is provided, the methodcomprising culturing T cells obtained from a blood sample from a subjectin the presence of an antibody, antigen binding fragment or polypeptideaccording to the present invention so as to expand the T cellpopulation, collecting expanded T cells, and administering the expandedT cells to a subject in need of treatment.

In methods of the present invention the antibody, antigen bindingfragment or polypeptide may be provided as a composition as describedherein.

In some embodiments the antibody may be one of clones 1B9, 1H9, 1H10,2C7, 2F4, 2G6, 1D9, 1F4, 2C8.

Description

Antibodies

Antibodies according to the present invention preferably bind to TIM-3(the antigen), preferably human or rhesus TIM-3, optionally with a Kd(s⁻¹) in the range 1.0×10⁻⁶ to 4.0×10⁻⁴.

In any aspect of the present invention the antibody preferablyspecifically binds TIM-3 (e.g. human or rhesus).

Antibodies according to the present invention may be provided inisolated form.

Antibodies according to the present invention may exhibit least one ofthe following properties:

-   -   a) binds to human TIM-3 with a Kd of 1×10⁻⁶ or less preferably        one of ≤1×10⁻⁵, ≤1×10⁻⁴, or 1×10⁻³;    -   b) is cytotoxic against TIM-3 expressing cells (antibody        dependent cell-mediated cytotoxicity, ADCC), e.g. TIM-3        expressing acute myeloid leukemia cells    -   c) increases T -cell proliferation in a Mixed Lymphocyte        Reaction (MLR) assay (e.g. see Bromelow et al J.Immunol Methods,        2001 Jan. 1; 247(1-2):1-8);    -   d) increases interferon-gamma production in an MLR assay; or    -   e) increases interleukin-2 (IL-2) secretion in an MLR assay.

By “antibody” we include a fragment or derivative thereof, or asynthetic antibody or synthetic antibody fragment.

In view of today's techniques in relation to monoclonal antibodytechnology, antibodies can be prepared to most antigens. Theantigen-binding portion may be a part of an antibody (for example a Fabfragment) or a synthetic antibody fragment (for example a single chainFv fragment [ScFv]). Suitable monoclonal antibodies to selected antigensmay be prepared by known techniques, for example those disclosed in“Monoclonal Antibodies: A manual of techniques”, H Zola (CRC Press,1988) and in “Monoclonal Hybridoma Antibodies: Techniques andApplications”, J G R Hurrell (CRC Press, 1982). Chimaeric antibodies arediscussed by Neuberger et al (1988, 8th International BiotechnologySymposium Part 2, 792-799).

Monoclonal antibodies (mAbs) are useful in the methods of the inventionand are a homogenous population of antibodies specifically targeting asingle epitope on an antigen.

Polyclonal antibodies are useful in the methods of the invention.Monospecific polyclonal antibodies are preferred. Suitable polyclonalantibodies can be prepared using methods well known in the art.

Antigen binding fragments of antibodies, such as Fab and Fab₂ fragmentsmay also be used/provided as can genetically engineered antibodies andantibody fragments. The variable heavy (V_(H)) and variable light(V_(L)) domains of the antibody are involved in antigen recognition, afact first recognised by early protease digestion experiments. Furtherconfirmation was found by “humanisation” of rodent antibodies. Variabledomains of rodent origin may be fused to constant domains of humanorigin such that the resultant antibody retains the antigenicspecificity of the rodent parented antibody (Morrison et al (1984) Proc.Natl. Acad. Sd. USA 81, 6851-6855).

That antigenic specificity is conferred by variable domains and isindependent of the constant domains is known from experiments involvingthe bacterial expression of antibody fragments, all containing one ormore variable domains. These molecules include Fab-like molecules(Better et al (1988) Science 240, 1041); Fv molecules (Skerra et al(1988) Science 240, 1038); single-chain Fv (ScFv) molecules where theV_(H) and V_(L) partner domains are linked via a flexible oligopeptide(Bird et al (1988) Science 242, 423; Huston et al (1988) Proc. Natl.Acad. Sd. USA 85, 5879) and single domain antibodies (dAbs) comprisingisolated V domains (Ward et al (1989) Nature 341, 544). A general reviewof the techniques involved in the synthesis of antibody fragments whichretain their specific binding sites is to be found in Winter & Milstein(1991) Nature 349, 293-299.

By “ScFv molecules” we mean molecules wherein the V_(H) and V_(L)partner domains are covalently linked, e.g. by a flexible oligopeptide.

Fab, Fv, ScFv and dAb antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of the said fragments.

Whole antibodies, and F(ab′)₂ fragments are “bivalent”. By “bivalent” wemean that the said antibodies and F(ab′)₂ fragments have two antigencombining sites. In contrast, Fab, Fv, ScFv and dAb fragments aremonovalent, having only one antigen combining site. Synthetic antibodieswhich bind to TIM-3 may also be made using phage display technology asis well known in the art.

Aspects of the present invention include bi-specific antibodies, e.g.composed of two different fragments of two different antibodies, suchthat the bi-specific antibody binds two types of antigen. One of theantigens is TIM-3, the bi-specific antibody comprising a fragment asdescribed herein that binds to TIM-3. The antibody may contain adifferent fragment having affinity for a second antigen, which may beany desired antigen, for example CD3 which has been used in cancerimmunotherapy to bind to cytotoxic cells, recruit and target them to thesite of a tumor. Techniques for the preparation of bi-specificantibodies are well known in the art, e.g. see Mueller, D et al., (2010Biodrugs 24 (2): 89-98), Wozniak-Knopp Get al., (2010 Protein Eng Des 23(4): 289-297. Baeuerle, PA et al., (2009 Cancer Res 69 (12): 4941-4944).

Accordingly, the present invention provides an antibody or antigenbinding fragment which is capable of binding to TIM-3, and which is abispecific antibody or a bispecific antigen binding fragment. In someembodiments, the bispecific antibody or bispecific antigen bindingfragment may be isolated.

In some embodiments, the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding fragment or a polypeptideaccording to the present invention.

In some embodiments, the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding domain capable of bindingto TIM-3, wherein the antigen binding domain which is capable of bindingto TIM-3 comprises or consists of an antigen binding fragment or apolypeptide according to the present invention.

In some embodiments the bispecific antibodies and bispecific antigenbinding fragments comprise an antigen binding domain capable of bindingto TIM-3, and an antigen binding domain capable of binding to anothertarget protein.

The antigen binding domain capable of binding to another target proteinmay be capable of binding to another protein other than TIM-3. In someembodiments, the target protein is a cell surface receptor. In someembodiments, the target protein is a cell surface receptor expressed onthe cell surface of immune cells. In some embodiments, the targetprotein is a cell surface receptor expressed on the cell surface of Tcells.

In some embodiments, the antigen binding domain capable of binding toanother target protein may be capable of binding to a T cell receptor(TCR) complex or a component thereof. In some embodiments, the antigenbinding domain may be capable of binding to CD3 or a CD3 polypeptide. Insome embodiments, the antigen binding domain may be capable of bindingto one or more of the CD3 polypeptides CD3γ, CD3δ, CD3ζ, or CD3ε. Insome embodiments the bispecific antibody is capable of directing T cellactivity (e.g. cytotoxic activity) against a TIM-3 expressing cell. Insome embodiments the bispecific antibody is a bispecific T-cell engagerantibody. In some embodiments the bispecific antibody or fragment iscapable of directing T cell activity (e.g. cytotoxic activity) against aTIM-3 expressing cell. That is, in some embodiments, T cell activity(e.g. cytotoxic activity) against a TIM-3 expressing cell is increasedin the presence of the bispecific antibody or fragment (e.g. relative toactivity against a TIM-3 expressing cell in the absence of thebispecific antibody or fragment). T cell activity against a TIM-3expressing cell can be determined in vitro by methods well known to theskilled person, for example by incubating T cells with TIM-3 expressingcells and measuring cell lysis as described herein.

In some embodiments, the bispecific antibody is provided as a fusionprotein of two single-chain variable fragments (scFV) format, comprisinga V_(H) and V_(L) of a TIM-3 binding antibody or antibody fragmentaccording to the present invention, and a V_(H) and V_(L) of an antibodyor antibody fragment capable of binding to CD3 or a CD3 polypeptide.

In some embodiments, the antigen binding domain for CD3 or a CD3polypeptide may comprise the CDRs, light and heavy chain variabledomains or other CD3- or CD3 polypeptide- binding fragment of e.g.anti-CD3 antibody clone OKT3 (eBioscience), clone CD3-12 (AbD Serotec),clone UCHT1 (Southern Biotech) clone SP7 (Thermo Scientific PierceAntibodies), clone SPV-T3b (Thermo Fisher Scientific), clone S4.1 (7D6)(Thermo Fisher Scientific), clone MEM-57 (AbD Serotec), clone 37895(Miltenyi Biotec), clone CA-3 (Abcam), clone 4D10A6 (Abbiotec), cloneHIT3a (Abbiotec), clone LT3 (Source BioScience), clone B-B11(MyBioSource.com), clone 17A2 (Novus Biologicals), clone BC3(BioLegend), clone HAM25-1352(MBL International), clone CA-3(Bosterbio), clone RBT-CD3 (Lifespan BioSciences), Ham25-1157 (MerckMillipore), clone CRIS-7 (Peninsula Laboratories International), clone5B2, clone 2Q1160 (Santa Cruz Biotechnology), clone M01, clone B1.1(Abnova Corporation), clone EP449E (BioGenex), clone 6B8D1G5, clone6B1C12F3 (Sino Biological), clone CL1297 (Atlas Antibodies), clone CC23(Creative Diagnostics), clone TR66 (Enzo Life Sciences), clone MEM-92(Cedarlane), clone EPR4516 (Origene Technologies), clone 3A12H2(Proteintech Group), clone 33-2A3 (ALPCO), clone E272 (Biocare Medical),clone SP162, clone MRQ-39 (Sigma Aldrich), or clone F7.2.38 (Dako).

In some embodiments, the target protein may be a member of the CD28family. In some embodiments, the target protein may be a member of theCD28 family such as PD-1 (CD279), LAG3 (CD223), ICOS (CD278), CTLA4(CD152), BTLA (CD272) or CD28.

In some particular embodiments, the bispecific antibody or bispecificantigen binding fragment comprises an antigen binding domain capable ofbinding to CD3 or a CD3 polypeptide, and an antigen binding domaincapable of binding to TIM-3 comprising at the CDRs, light and heavychain variable domains or other TIM-3 binding fragment of clone 2C7described herein.

In some embodiments, the bispecific antibody of the present inventionmay exhibit least one of the following properties:

-   -   a) increases or enhances cell killing (e.g. T cell mediated cell        killing) of TIM-3 expressing cells (antibody dependent        cell-mediated cytotoxicity, ADCC), e.g. TIM-3 expressing acute        myeloid leukemia cells;    -   b) increases or enhances cell killing (e.g. T cell mediated cell        killing) of TIM-3 expressing stem cells (antibody dependent        cell-mediated cytotoxicity, ADCC), e.g. TIM-3 expressing,        CD34+acute myeloid leukemia cells;

In some embodiments, the antigen binding domain for PD-1 may comprisethe CDRs, light and heavy chain variable domains or other PD-1 bindingfragment of e.g. anti-PD-1 antibody clone J116, clone MIH4(eBioscience), clone 7A11B1 (Rockland Immunochemicals Inc.), clone192106 (R&D Systems), clone J110, clone J105 (MBL International), clone12A7D7, clone 7A11B1 (Abbiotec), clone #9X21 (MyBioSource.com), clone4H4D1 (Proteintech Group), clone D3W4U, clone D3O4S (Cell SignalingTechnology), clone RMP1-30, clone RMP1-14 (Merck Millipore), cloneEH12.2H7 (BioLegend), clone 10B1227 (United States Biological), cloneUMAB198, or clone UMAB197 (Origene Technologies). In some embodiments,the antigen binding domain for LAG3 may comprise the CDRs, light andheavy chain variable domains or other LAG3 binding fragment of e.g.anti-LAG3 antibody clone 17B4 (Enzo Life Sciences), clone 333210 (R&DSystems), or clone 14L676 (United States Biological). In someembodiments, the antigen binding domain for ICOS may comprise the CDRs,light and heavy chain variable domains or other ICOS binding fragment ofe.g. anti-ICOS antibody clone ISA-3 (eBioscience), clone SP98 (NovusBiologicals), clone 1G1, clone 3G4 (Abnova Corporation), clone 669222(R&D Systems), clone TQ09 (Creative Diagnostics), or clone C398.4A(BioLegend). In some embodiments, the antigen binding domain for CTLA4may comprise the CDRs, light and heavy chain variable domains or otherCTLA4 binding fragment of e.g. anti-CTLA4 antibody clone 2F1, clone 1 F4(Abnova Corporation), clone 9H10 (EMD Millipore), clone BNU3 (GeneTex),clone 1E2, clone AS32 (LifeSpan BioSciences) clone A3.4H2.H12 (AcrisAntibodies), clone 060 (Sino Biological), clone BU5G3 (CreativeDiagnostics), clone MIH8 (MBL International), clone A3.6B10.G1, or cloneL3D10 (BioLegend). In some embodiments, the antigen binding domain forBTLA may comprise the CDRs, light and heavy chain variable domains orother BTLA binding fragment of e.g. anti-BTLA antibody clone 1B7, clone2G8, clone 4C5 (Abnova Corporation), clone 4B8 (antibodies-online),clone MIH26 (Thermo Scientific

Pierce Antibodies), clone UMAB61 (OriGene Technologies), clone 330104(R&D Systems), clone 1B4 (LifeSpan BioSciences), clone 440205, clone 5E7(Creative Diagnostics). In some embodiments, the antigen binding domainfor CD28 may comprise the CDRs, light and heavy chain variable domainsor other CD28 binding fragment of e.g. anti-CD28 antibody clone CD28.6(eBioscience), clone CD28.2, clone JJ319 (Novus Biologicals), clone204.12, clone B-23, clone 10F3 (Thermo Scientific Pierce Antibodies),clone 37407 (R&D Systems), clone 204-12 (Abnova Corporation), clone 15E8(EMD Millipore), clone 204-12, clone YTH913.12 (AbD Serotec), clone B-T3(Acris Antibodies), clone 9H6E2 (Sino Biological), clone C28/77(MyBioSource.com), clone KOLT-2 (ALPCO), clone 152-2E10 (Santa CruzBiotechnology), or clone XPH-56 (Creative Diagnostics).

An antigen binding domain of a bispecific antibody or bispecific antigenbinding fragment according to the present invention may be any domain ofa polypeptide which is capable of binding to an antigen. In someembodiments, an antigen binding domain comprises at least the threelight chain CDRs (i.e. LC-CDR1, LC-CDR2 and LC-CDR3) and three heavychain CDRs (i.e. HC-CDR1, HC-CDR2 and HC-CDR3) which together define theantigen binding region of an antibody or antigen binding fragment. Insome embodiments, an antigen binding domain may comprise the light chainvariable domain and heavy chain variable domain of an antibody orantigen binding fragment. In some embodiments, an antigen binding domainmay comprise the light chain polypeptide and heavy chain polypeptide ofan antibody or antigen binding fragment.

Bispecific antibodies and bispecific antigen binding fragments accordingto the invention may be provided in any suitable format, such as thoseformats described in Kontermann MAbs 2012, 4(2): 182-197, which ishereby incorporated by reference in its entirety. For example, abispecific antibody or bispecific antigen binding fragment may be abispecific antibody conjugate (e.g. an IgG2, F(ab′)₂ or CovX-Body), abispecific IgG or IgG-like molecule (e.g. an IgG, scFv₄-Ig, IgG-scFv,scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, 2 in 1-IgG, mAb², or Tandemab commonLC), an asymmetric bispecific IgG or IgG-like molecule (e.g. a kih IgG,kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair orSEED-body), a small bispecific antibody molecule (e.g. a Diabody (Db),dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb/VHH, triplebody, triple head, Fab-scFv, or F(ab′)₂-scFv₂), a bispecific Fc andC_(H)3 fusion protein (e.g. a taFv-Fc, Di-diabody, scDb-C_(H)3,scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-C_(H)3), or abispecific fusion protein (e.g. a scFv₂-albumin, scDb-albumin,taFv-toxin, DNL-Fab₃, DNL-Fab₄-IgG, DNL-Fab₄-IgG-cytokine₂). See inparticular FIG. 2 of Kontermann MAbs 2012, 4(2): 182-19.

The skilled person is able to design and prepare bispecific antibodiesand bispecific antigen binding fragments according to the presentinvention. Methods for producing bispecific antibodies includechemically crosslinking of antibodies or antibody fragments, e.g. withreducible disulphide or non-reducible thioether bonds, for example asdescribed in Segal and Bast, 2001. Production of Bispecific Antibodies.Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16, which ishereby incorporated by reference in its entirety. For example,N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used tochemically crosslink e.g. Fab fragments via hinge region SH— groups, tocreate disulfide-linked bispecific F(ab)₂ heterodimers.

Other methods for producing bispecific antibodies include fusingantibody-producing hybridomas e.g. with polyethylene glycol, to producea quadroma cell capable of secreting bispecific antibody, for example asdescribed in D. M. and Bast, B. J. 2001. Production of BispecificAntibodies. Current Protocols in Immunology. 14:IV:2.13:2.13.1-2.13.16.

Bispecific antibodies and bispecific antigen binding fragments accordingto the present invention can also be produced recombinantly, byexpression from e.g. a nucleic acid construct encoding polypeptides forthe antigen binding molecules, for example as described in AntibodyEngineering: Methods and Protocols, Second Edition (Humana Press, 2012),at Chapter 40: Production of Bispecific Antibodies: Diabodies and TandemscFv (Hornig and Färber-Schwarz), or French, How to make bispecificantibodies,

Methods Mol. Med. 2000; 40:333-339, the entire contents of both of whichare hereby incorporated by reference.

For example, a DNA construct encoding the light and heavy chain variabledomains for the two antigen binding domains (i.e. the light and heavychain variable domains for the antigen binding domain capable of bindingTIM-3, and the light and heavy chain variable domains for the antigenbinding domain capable of binding to another target protein), andincluding sequences encoding a suitable linker or dimerization domainbetween the antigen binding domains can be prepared by molecular cloningtechniques. Recombinant bispecific antibody can thereafter be producedby expression (e.g. in vitro) of the construct in a suitable host cell(e.g. a mammalian host cell), and expressed recombinant bispecificantibody can then optionally be purified.

Antibodies, antigen fragments or polypeptides according to the presentinvention may also be used to construct chimeric antigen receptors (CAR;also called artificial T-cell receptors) in which a receptor isengineered by recombinant techniques to graft a selected specificityonto an immune cell. For example, the specificity of a monoclonalantibody may be grafted onto a T-cell, and the modified T-cells may finduse in treatment of disease, e.g. cancer. One form of CAR is a fusion ofan scFv comprising an antibody, antigen fragment or polypeptideaccording to the present invention to a transmembrane and endo domain ofa suitable receptor scaffold. Techniques for the generation of CARs aredescribed in Pule, M et al., (2003 Cytotherapy 5 (3): 211-26).

Antibodies may be produced by a process of affinity maturation in whicha modified antibody is generated that has an improvement in the affinityof the antibody for antigen, compared to an unmodified parent antibody.Affinity-matured antibodies may be produced by procedures known in theart, e.g., Marks et al., Rio/Technology 10:779-783 (1992); Barbas et al.Proc Nat. Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995);Jackson et al., J. Immunol. 154(7):331 0-15 9 (1995); and Hawkins et al,J. Mol. Biol. 226:889-896 (1992).

Antibodies according to the present invention preferably exhibitspecific binding to TIM-3. An antibody that specifically binds to atarget molecule preferably binds the target with greater affinity,and/or with greater duration than it binds to other targets. In oneembodiment, the extent of binding of an antibody to an unrelated targetis less than about 10% of the binding of the antibody to the target asmeasured, e.g., by ELISA, or by a radioimmunoassay (RIA). Alternatively,the binding specificity may be reflected in terms of binding affinitywhere the anti-TIM-3 antibody of the present invention binds to TIM-3with a Kd that is at least 0.1 order of magnitude (i.e. 0.1×10^(n),where n is an integer representing the order of magnitude) greater thanthe Kd of the antibody towards another target molecule, e.g. anothermember of the TIM-3 family. This may optionally be one of at least 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0.

Antibodies according to the present invention preferably have adissociation constant (Kd) of one of ≤1×10⁻⁶, ≤1×10⁻⁵, ≤1×10⁻⁴, or≤1×10⁻³. Binding affinity of an antibody for its target is oftendescribed in terms of its dissociation constant (Kd). Binding affinitycan be measured by methods known in the art, such as by Surface PlasmonResonance (SPR), or by a radiolabeled antigen binding assay (RIA)performed with the Fab version of the antibody and antigen molecule.

Antibodies according to the present invention may be “antagonist”antibodies that inhibit or reduce a biological activity of the antigento which it binds. Blocking of TIM-3 assists in the restoration ofT-cell function by inhibiting the immune-inhibitory signalling pathwaymediated by TIM-3.

In some aspects, the antibody is 169, or a variant of 169. 1 B9comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 12) EAWDYYVAAGYHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 32)GYVAGSDA

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 1 H9, or a variant of 1 H9. 1 H9comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 13) DSWDSADASGVHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 33)GYVAGFDD

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 1H10, or a variant of 1H10. 1H10comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 14) DSWDYDYAAGVHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 34)GYVAGFDS

GDR sequences determined by Kabat definition.

In some aspects, the antibody is 2C7, or a variant of 2C7. 2C7 comprisesthe following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 15) DSWDSYLAAGVHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 35)GYVAGYDY

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 2F4, or a variant of 2F4. 2F4 comprisesthe following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 16) ESWDYDYASGVHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 36)GYVAGSDA

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 2G6, or a variant of 2G6. 2G6 comprisesthe following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 17) DSWDSSDSSGVHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 37)GYVAGFDS

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 1 D9, or a variant of 1 D9. 1D9comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYV LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 18) EAWDSAYAAGSHeavy chain: HC-CDR1: (SEQ ID NO: 30)  GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 38)GYYAGDDY

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 1 F4, or a variant of 1 F4. 1F4comprises the following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 19) DSWDAALSAGVHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 39)GYVAGYDY

CDR sequences determined by Kabat definition.

In some aspects, the antibody is 2C8, or a variant of 2C8. 2C8 comprisesthe following CDR sequences:

Light chain: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 20) ESWDAAAAAGYHeavy chain: HC-CDR1: (SEQ ID NO: 30) GGSFSGYYWS, or (SEQ ID NO: 61)GYYWS HC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 40)GYVAGSDA

CDR sequences determined by Kabat definition.

Antibodies according to the present invention may comprise the CDRs ofone of clones 1B9, 1H9, 1H10, 2C7, 2F4, 2G6, 1D9, 1F4, 2C8 or one of SEQID NOs 1 and 21, 2 and 22, 3 and 23, 4 and 24, 5 and 25, 6 and 26, 7 and27, 8 and 28 or 9 and 29. In an antibody according to the presentinvention one or two or three or four of the six CDR sequences may vary.A variant may have one or two amino acid substitutions in one or two ofthe six CDR sequences.

Amino acid sequences of the V_(H) and V_(L) chains of ant-TIM-3 clonesare shown in FIGS. 1 and 2. The encoding nucleotide sequences are shownin FIG. 4.

The light and heavy chain CDRs may also be particularly useful inconjunction with a number of different framework regions. Accordingly,light and/or heavy chains having LC-CDR1-3 or HC-CDR1-3 may possess analternative framework region. Suitable framework regions are well knownin the art and are described for example in M. Lefranc & G. Le:franc(2001) “The Immunoglobulin FactsBook”, Academic Press, incorporatedherein by reference.

In this specification, antibodies may have V_(H) and/or VL chainscomprising an amino acid sequence that has a high percentage sequenceidentity to one or more of the V_(H) and/or V_(L) amino acid sequencesof SEQ ID NOs 1 and 21, 2 and 22, 3 and 23, 4 and 24, 5 and 25, 6 and26, 7 and 27, 8 and 28 or 9 and 29 or to one or the amino acid sequencesshown in FIGS. 1 and 2.

For example, antibodies according to the present invention includeantibodies that bind TIM-3 and have a V_(H) chain that comprises anamino acid sequence having at least 70%, more preferably one of at least75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, sequence identity to the V_(H) chain amino acidsequence of one of SEQ ID NOs 1 and 21, 2 and 22, 3 and 23, 4 and 24, 5and 25, 6 and 26, 7 and 27, 8 and 28 or 9 and 29 or to one or the aminoacid sequences shown in FIGS. 1 and 2.

Antibodies according to the present invention may be detectably labelledor, at least, capable of detection. For example, the antibody may belabelled with a radioactive atom or a coloured molecule or a fluorescentmolecule or a molecule which can be readily detected in any other way.Suitable detectable molecules include fluorescent proteins, luciferase,enzyme substrates, and radiolabels. The binding moiety may be directlylabelled with a detectable label or it may be indirectly labelled. Forexample, the binding moiety may be an unlabelled antibody which can bedetected by another antibody which is itself labelled. Alternatively,the second antibody may have bound to it biotin and binding of labelledstreptavidin to the biotin is used to indirectly label the firstantibody.

Methods of Detection

Antibodies, or antigen binding fragments, described herein may be usedin methods that involve the binding of the antibody or antigen bindingfragment to TIM-3. Such methods may involve detection of the boundcomplex of antibody, or antigen binding fragment, and TIM-3. As such, inone embodiment a method is provided, the method comprising contacting asample containing, or suspected to contain, TIM-3 with an antibody orantigen binding fragment as described herein and detecting the formationof a complex of antibody, or antigen binding fragment, and TIM-3.

Suitable method formats are well known in the art, includingimmunoassays such as sandwich assays, e.g. ELISA. The method may involvelabelling the antibody, or antigen binding fragment, or TIM-3, or both,with a detectable label, e.g. fluorescent, luminescent or radio-label.

Methods of this kind may provide the basis of a method of diagnosis of adisease or condition requiring detection and or quantitation of TIM-3.Such methods may be performed in vitro on a patient sample, or followingprocessing of a patient sample. Once the sample is collected, thepatient is not required to be present for the in vitro method ofdiagnosis to be performed and therefore the method may be one which isnot practised on the human or animal body.

Such methods may involve determining the amount of TIM-3 present in apatient sample. The method may further comprise comparing the determinedamount against a standard or reference value as part of the process ofreaching a diagnosis. Other diagnostic tests may be used in conjunctionwith those described here to enhance the accuracy of the diagnosis orprognosis or to confirm a result obtained by using the tests describedhere.

The level of TIM-3 present in a patient sample may be indicative that apatient may respond to treatment with an anti-TIM-3 antibody. Thepresence of a high level of TIM-3 in a sample may be used to select apatient for treatment with an anti-TIM-3 antibody. The antibodies of thepresent invention may therefore be used to select a patient fortreatment with anti-TIM-3 therapy.

Detection in a sample of TIM-3 may be used for the purpose of diagnosisof a T-cell dysfunctional disorder or a cancerous condition in thepatient, diagnosis of a predisposition to a cancerous condition or forproviding a prognosis (prognosticating) of a cancerous condition. Thediagnosis or prognosis may relate to an existing (previously diagnosed)cancerous condition, which may be benign or malignant, may relate to asuspected cancerous condition or may relate to the screening forcancerous conditions in the patient (which may be previouslyundiagnosed).

In one embodiment the level of TIM3 expression on CD8+ T cells may bedetected in order to indicate the degree of T-cell exhaustion andseverity of the disease state. In some cases, the level of TIM-3expression on T cells or tumor cells may be used to select a patient fortreatment with a modulator of TIM3 signalling, such as an anti-TIM3antibody or anti-TIM3 agent.

A sample may be taken from any tissue or bodily fluid. The sample maycomprise or may be derived from: a quantity of blood; a quantity ofserum derived from the individual's blood which may comprise the fluidportion of the blood obtained after removal of the fibrin clot and bloodcells; a tissue sample or biopsy; or cells isolated from saidindividual.

Methods according to the present invention are preferably performed invitro. The term “in vitro” is intended to encompass experiments withcells in culture whereas the term “in vivo” is intended to encompassexperiments with intact multi-cellular organisms.

Therapeutic Applications

Antibodies, antigen binding fragments and polypeptides according to thepresent invention and compositions comprising such agents may beprovided for use in methods of medical treatment. Treatment may beprovided to subjects having a disease or condition in need of treatment.The disease or condition may be one of a T-cell dysfunctional disorder,including a T-cell dysfunctional disorder associated with a cancer, acancer, or an infectious disease

A T-cell dysfunctional disorder may be a disease or condition in whichnormal T-cell function is impaired causing downregulation of thesubject's immune response to pathogenic antigens, e.g. generated byinfection by exogenous agents such as microorganisms, bacteria andviruses, or generated by the host in some disease states such as in someforms of cancer (e.g. in the form of tumor associated antigens).

The T-cell dysfunctional disorder may comprise T-cell exhaustion orT-cell anergy. T-cell exhaustion comprises a state in which CD8+ T-cellsfail to proliferate or exert T-cell effector functions such ascytotoxicity and cytokine (e.g. IFNγ) secretion in response to antigenstimulation. Exhausted T-cells may also be characterised by sustainedupregulation of TIM-3, where blockade of TIM-3:galectin 9 interactionsmay reverse the T-cell exhaustion and restore antigen-specific T cellresponses.

The T-cell dysfunctional disorder may be manifest as an infection, orinability to mount an effective immune response against an infection.The infection may be chronic, persistent, latent or slow, and may be theresult of bacterial, viral, fungal or parasitic infection. As such,treatment may be provided to patients having a bacterial, viral orfungal infection. Examples of bacterial infections include infectionwith Helicobacter pylori. Examples of viral infections include infectionwith HIV, hepatitis B or hepatitis C.

The T-cell dysfunctional disorder may be associated with a cancer, suchas tumor immune escape. Many human tumors express tumor-associatedantigens recognised by T cells and capable of inducing an immuneresponse. However, immune evasion is common and is believed to bemediated by a number of soluble factors, including galectin 9. As such,blocking the interaction of TIM-3 and galectin 9 may inhibit thisnegative immunoregulatory signal to tumor cells and enhancetumor-specific CD8⁺ T-cell immunity.

Cancers may also be treated where there is no indication of a T-celldysfunctional disorder such as T-cell exhaustion. Antibodies accordingto the present invention may be cytotoxic against TIM-3 expressingcells, such as T-cells, e.g. exhausted T-cells or cancer cells such asacute myeloid leukemia cells. As such, the antibody, antigen bindingfragment or polypeptides described herein may be useful in methodsinvolving antibody dependent cell-mediated cytotoxicity (ADCC) orcomplement dependent cytotoxicity (CDC), or in any method that recruitsimmune effector functions to kill the target cells, sucha as CAR cells,or bispecific antibodies targeting CD3.

The use of an antibody, antigen binding fragment or polypeptideaccording to the present invention allows the subject to suppress TIM-3signalling and mount an effective immune response with limitedimpairment, evasion or induction of tumor immune escape. In suchtreatments, the antibody, antigen binding fragment or polypeptide mayprovide a treatment for cancer that involves prevention of thedevelopment of tumor immune escape.

The treatment may be aimed at prevention of the T-cell dysfunctionaldisorder, e.g. prevention of infection or of the development orprogression of a cancer. As such, the antibodies, antigen bindingfragments and polypeptides may be used to formulate pharmaceuticalcompositions or medicaments and subjects may be prophylactically treatedagainst development of a disease state. This may take place before theonset of symptoms of the disease state, and/or may be given to subjectsconsidered to be at greater risk of infection or development of cancer.

Treatment may comprise co-therapy with a vaccine, e.g. T-cell vaccine,which may involve simultaneous, separate or sequential therapy, orcombined administration of vaccine and antibody, antigen bindingfragment or polypeptide in a single composition. In this context, theantibody, antigen binding fragment or polypeptide may be provided as anadjuvant to the vaccine. Limited proliferative potential of exhausted Tcells has been attributed as a main reason for failure of T-cellimmunotherapy and combination an agent capable of blocking or reversingT cell exhaustion is a potential strategy for improving the efficacy ofT-cell immunotherapy (Barber et al., Nature Vol 439, No. 9 p682-687February 2006).

Administration of an antibody, antigen binding fragment or polypeptideis preferably in a “therapeutically effective amount”, this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of the disease being treated. Prescription oftreatment, e.g. decisions on dosage etc., is within the responsibilityof general practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

Formulating Pharmaceutically Useful Compositions and Medicaments

Antibodies, antigen binding fragments and polypeptides according to thepresent invention may be formulated as pharmaceutical compositions forclinical use and may comprise a pharmaceutically acceptable carrier,diluent, excipient or adjuvant.

In accordance with the present invention methods are also provided forthe production of pharmaceutically useful compositions, such methods ofproduction may comprise one or more steps selected from: isolating anantibody, antigen binding fragment or polypeptide as described herein;and/or mixing an isolated antibody, antigen binding fragment orpolypeptide as described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

For example, a further aspect of the present invention relates to amethod of formulating or producing a medicament or pharmaceuticalcomposition for use in the treatment of a T-cell dysfunctional disorder,the method comprising formulating a pharmaceutical composition ormedicament by mixing an antibody, antigen binding fragment orpolypeptide as described herein with a pharmaceutically acceptablecarrier, adjuvant, excipient or diluent.

Infection

An infection may be any infection or infectious disease, e.g. bacterial,viral, fungal, or parasitic infection. In some embodiments it may beparticularly desirable to treat chronic/persistent infections, e.g.where such infections are associated with T cell dysfunction or T cellexhaustion.

It is well established that T cell exhaustion is a state of T celldysfunction that arises during many chronic infections (including viral,bacterial and parasitic), as well as in cancer (Wherry Nature ImmunologyVol.12, No.6, p492-499, June 2011).

TIM-3 expression has been reported to play an important pathogenic rolein patients having chronic infection (e.g. as reported by Golden-MasonL, et al., J Virol. 2009; 83(18):9122-9130.)

Examples of bacterial infections that may be treated include infectionby Bacillus spp., Bordetella pertussis, Clostridium spp.,Corynebacterium spp., Vibrio chloerae, Staphylococcus spp.,Streptococcus spp. Escherichia, Klebsiella, Proteus, Yersinia, Erwina,Salmonella, Listeria sp, Helicobacter pylori, mycobacteria (e.g.Mycobacterium tuberculosis) and Pseudomonas aeruginosa. For example, thebacterial infection may be sepsis or tuberculosis.

Yao et al (PD-1 on dendritic cells impedes innate immunity againstbacterial infection. Blood 113(23):5811-5818 Jun. 4 2009) establishedPD-1 in the negative regulation of DC function during innate immuneresponse to infection by Listeria monocytogenes. Brahmamdam et al(Delayed administration of anti-PD-1 antibody reverses immunedysfunction and improves survival during sepsis. Journal of LeukocyteBiology vo.88, no.2 233-240, August 2010) reported that anti-PD-1antibody administered 24 h after sepsis prevented sepsis-induceddepletion of lymphocytes and DCs, increased Bcl-xL, blocked apoptosisand improved survival. Tim3:Galectin-9 interactions have been reportedto mediate T cell exhaustion and mediate the innate and adaptive immuneresponse to infection by Mycobacterium tuberculosis (Jayaraman et al.,The Journal of Immunology 2012, 188, 70.6).

Examples of viral infections that may be treated include infection byinfluenza virus, measles virus, hepatitis B virus (HBV), hepatitis Cvirus (HCV), human immunodeficiency virus (HIV), lymphocyticchoriomeningitis virus (LCMV), Herpes simplex virus and human papillomavirus.

Chronic viral infections, such as those caused by HCV, HBV, and HIVcommonly involve mechanisms to evade immune clearance. Expression ofPD-1 and TIM-3 have been identified as correlating with defective T cellresponses to hepatitis C virus (HCV) (McMahan et al., The Journal ofClinical Investigation Vol. 120, No. 12 p4546-4557, December 2010). InHCV, McMahan et al (supra) found that the level of dual TIM-3 and PD-1expression on HCV-specific CTLs predated the development of viralpersistence, providing prognostic information. Barber et al. (Nature Vol439, No. 9 p682-687 Feb 2006) reported that PD-1 is upregulated duringchronic viral infection. In mice infected with LCMV they reported thatblockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect onCD8 T cells, restoring their ability to undergo proliferation, secretecytokines, kill infected cells and decrease viral load. PD-1 is alsoupregulated in HIV infection (Said et al., Nature Medicine Vol. 16, No.4p452-460 April 2010). Blocking interaction between PD-1 and PD-L1contributed to viral clearance and improved T cell function in animalmodels of chronic viral infection (Said et al., supra).

Examples of fungal infections that may be treated include infection byAlternaria sp, Aspergillus sp, Candida sp and Histoplasma sp. The fungalinfection may be fungal sepsis or histoplasmosis.

Chang et al (Blockade of the negative co-stimulatory molecules PD-1 andCTLA-4 improves survival in primary and secondary fungal sepsis.Critical Care 2013, 17:R85) reported that anti-PD1 antibodies werehighly effective at improving survival in primary and secondary fungalsepsis. Lázár-Molnár et al (The PD-1/PD-L costimulatory pathwaycritically affects host resistance to the pathogenic fungus Histoplasmacapsulatum PNAS vol. 105, no.7, p2658-2663, 19 Feb. 2008) reported thatanti-PD-1 antibody significantly increased survival of mice infectedwith Histoplasma capsulatum. As such, the importance of T cellexhaustion in mediating fungal infection is well established. Examplesof parasitic infections that may be treated include infection byPlasmodium species (e.g. Plasmodium falciparum, Plasmodium yoeli,Plasmodium ovale, Plasmodium vivax, or Plasmodium chabaudi chabaudi).The parasitic infection may be a disease such as malaria, leishmaniasisand toxoplasmosis.

Infection of humans with Plasmodium falciparum has been shown to resultin higher expression of PD-1 and T cell exhaustion mice (Butler et al.,Nature Immunology Vol.13, No.12, p 188-195 February 2012). Blockade ofPD-L1 and LAG-3 using anti-PD-L1 and anti-LAG-3 monoclonal antibodies invivo contributed to the restoration of CD4⁺ T-cell function,amplification of the number of follicular helper T cells,germinal-center B cells and plasmablasts, enhanced protective antibodiesand rapidly cleared blood-stage malaria in mice. It was also shown toblock the development of chronic infection (Butler et al.,supra).

Cancer

A cancer may be any unwanted cell proliferation (or any diseasemanifesting itself by unwanted cell proliferation), neoplasm or tumor orincreased risk of or predisposition to the unwanted cell proliferation,neoplasm or tumor. The cancer may be benign or malignant and may beprimary or secondary (metastatic). A neoplasm or tumor may be anyabnormal growth or proliferation of cells and may be located in anytissue. Examples of tissues include the adrenal gland, adrenal medulla,anus, appendix, bladder, blood, bone, bone marrow, brain, breast, cecum,central nervous system (including or excluding the brain) cerebellum,cervix, colon, duodenum, endometrium, epithelial cells (e.g. renalepithelia), gallbladder, oesophagus, glial cells, heart, ileum, jejunum,kidney, lacrimal glad, larynx, liver, lung, lymph, lymph node,lymphoblast, maxilla, mediastinum, mesentery, myometrium, nasopharynx,omentume, oral cavity, ovary, pancreas, parotid gland, peripheralnervous system, peritoneum, pleura, prostate, salivary gland, sigmoidcolon, skin, small intestine, soft tissues, spleen, stomach, testis,thymus, thyroid gland, tongue, tonsil, trachea, uterus, vulva, whiteblood cells.

Tumors to be treated may be nervous or non-nervous system tumors.Nervous system tumors may originate either in the central or peripheralnervous system, e.g. glioma, medulloblastoma, meningioma, neurofibroma,ependymoma, Schwannoma, neurofibrosarcoma, astrocytoma andoligodendroglioma. Non-nervous system cancers/tumors may originate inany other non-nervous tissue, examples include melanoma, mesothelioma,lymphoma, myeloma, leukemia, Non-Hodgkin's lymphoma (NHL), Hodgkin'slymphoma, chronic myelogenous leukemia (CML), acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), cutaneous T-cell lymphoma (CTCL),chronic lymphocytic leukemia (CLL), hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer, lung cancer , colon cancer, ovariancancer, pancreatic cancer, thymic carcinoma, NSCLC, haematologic cancerand sarcoma.

Adoptive T Cell Transfer Therapy

Adoptive T cell transfer therapy generally refers to a process in whichwhite blood cells are removed from a subject, typically by drawing ablood sample from which white blood cells are separated, expanded invitro or ex vivo and returned either to the same subject or to adifferent subject. The treatment is typically aimed at increasing theamount/concentration of an active form of the required T cell populationin the subject. Such treatment may be beneficial in subjectsexperiencing T cell exhaustion.

Antibodies capable of blocking the mechanism of T cell exhaustion, orreversing it, provide a means of enhancing T cell activity and promotingT cell expansion.

Accordingly, in a further aspect of the present invention a method isprovided for expanding a population of T cells, wherein T cells arecontacted in vitro or ex vivo with an antibody, antigen binding fragmentor polypeptide according to the present invention.

The method may optionally comprise one or more of the following steps:taking a blood sample from a subject; isolating T cells from the bloodsample; culturing the T cells in in vitro or ex vivo cell culture (wherethey may be contacted with the antibody, antigen binding fragment orpolypeptide), collecting an expanded population of T cells; mixing the Tcells with an adjuvant, diluent, or carrier; administering the expandedT cells to a subject.

Accordingly, in some aspects of the present invention a method oftreatment of a subject having a T-cell dysfunctional disorder isprovided, the method comprising obtaining a blood sample from a subjectin need of treatment, culturing T cells obtained from the blood samplein the presence of an antibody, antigen binding fragment or polypeptideaccording to the present invention so as to expand the T cellpopulation, collecting expanded T cells, and administering the expandedT cells to a subject in need of treatment.

The T cells may be obtained from a subject requiring treatment, and maybe isolated and/or purified. They may be a CD4⁺ and/or CD8⁺ T-cellpopulation. The T-cells may represent a population experiencing T cellexhaustion and may optionally have upregulated expression of TIM-3.

During culture, T cells may be contacted with the antibody, antigenbinding fragment or polypeptide under conditions and for a period oftime suitable to allow expansion of the T cells to a desired number ofcells. After a suitable period of time the T cells may be harvested,optionally concentrated, and may be mixed with a suitable carrier,adjuvant or diluent and returned to the subject's body. A subject mayundergo one or more rounds of such therapy.

Methods of T cell expansion are well known in the art, such as thosedescribed in Kalamasz et al., J lmmunother 2004 September-October;27(5):405-18; Montes et al., Clin Exp Immunol 2005 November;142(2):292-302; Wölfl and Greenburg Nature Protocols 9 p950-966 27 Mar.2014; Trickett and Kwan Journal of Immunological Methods Vol. 275,Issues 1-2, 1 Apr. 2003, p251-255; Butler et al PLoSONE 7(1) 12 Jan.2012.

Simultaneous or Sequential Administration

Compositions may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

In this specification an antibody, antigen binding fragment orpolypeptide of the present invention and an anti-infective agent orchemotherapeutic agent (therapeutic agent) may be administeredsimultaneously or sequentially.

In some embodiments, treatment with an antibody, antigen bindingfragment or polypeptide of the present invention may be accompanied bychemotherapy.

Simultaneous administration refers to administration of the antibody,antigen binding fragment or polypeptide and therapeutic agent together,for example as a pharmaceutical composition containing both agents(combined preparation), or immediately after each other and optionallyvia the same route of administration, e.g. to the same artery, vein orother blood vessel.

Sequential administration refers to administration of one of theantibody, antigen binding fragment or polypeptide or therapeutic agentfollowed after a given time interval by separate administration of theother agent. It is not required that the two agents are administered bythe same route, although this is the case in some embodiments. The timeinterval may be any time interval.

Anti-Infective Agents

In treating infection, an antibody, antigen binding fragment orpolypeptide of the present invention may be administered in combinationwith an anti-infective agent, as described above. The anti-infectiveagent may be an agent known to have action against the microorganism orvirus responsible for the infection.

Suitable anti-infective agents include antibiotics (such as penicillins,cephalosporins, rifamycins, lipiarmycins, quinolones, sulfonamides,macrolides, lincosamides, tetracyclines, cyclic lipopeptides,glycylcyclines, oxazolidinones, and lipiarmycins), anti-viral agents(such as reverse transcriptase inhibitors, integrase inhibitors,transcription factor inhibitors, antisense and siRNA agents and proteaseinhibitors), anti-fungal agents (such as polyenes, imidiazoles,triazoles, thiazoles, allylamines, and echinocandins) and anti-parasiticagents (such as antinematode agents, anticestode agents, antitrematodeagents, antiamoebic agents and antiprotozoal agents).

Chemotherapy

Chemotherapy refers to treatment of a cancer with a drug or withionising radiation (e.g. radiotherapy using X-rays or y-rays). Inpreferred embodiments chemotherapy refers to treatment with a drug. Thedrug may be a chemical entity, e.g. small molecule pharmaceutical,antibiotic, DNA intercalator, protein inhibitor (e.g. kinase inhibitor),or a biological agent, e.g. antibody, antibody fragment, nucleic acid orpeptide aptamer, nucleic acid (e.g. DNA, RNA), peptide, polypeptide, orprotein. The drug may be formulated as a pharmaceutical composition ormedicament. The formulation may comprise one or more drugs (e.g. one ormore active agents) together with one or more pharmaceuticallyacceptable diluents, excipients or carriers.

A treatment may involve administration of more than one drug. A drug maybe administered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated. For example, the chemotherapy may be a co-therapy involvingadministration of two drugs, one or more of which may be intended totreat the cancer.

The chemotherapy may be administered by one or more routes ofadministration, e.g. parenteral, intravenous injection, oral,subcutaneous, intradermal or intratumoral.

The chemotherapy may be administered according to a treatment regime.The treatment regime may be a pre-determined timetable, plan, scheme orschedule of chemotherapy administration which may be prepared by aphysician or medical practitioner and may be tailored to suit thepatient requiring treatment.

The treatment regime may indicate one or more of: the type ofchemotherapy to administer to the patient; the dose of each drug orradiation; the time interval between administrations; the length of eachtreatment; the number and nature of any treatment holidays, if any etc.For a co-therapy a single treatment regime may be provided whichindicates how each drug is to be administered.

Chemotherapeutic drugs and biologics may be selected from:

-   -   alkylating agents such as cisplatin, carboplatin,        mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide;    -   purine or pyrimidine anti-metabolites such as azathiopurine or        mercaptopurine;    -   alkaloids and terpenoids, such as vinca alkaloids (e.g.        vincristine, vinblastine, vinorelbine, vindesine),        podophyllotoxin, etoposide, teniposide, taxanes such as        paclitaxel (Taxol™), docetaxel;    -   topoisomerase inhibitors such as the type I topoisomerase        inhibitors camptothecins irinotecan and topotecan, or the type        II topoisomerase inhibitorsamsacrine, etoposide, etoposide        phosphate, teniposide;    -   antitumor antibiotics (e.g. anthracyline antibiotics) such as        dactinomycin, doxorubicin (Adriamycin™), epirubicin, bleomycin,        rapamycin;    -   antibody based agents, such as anti-PD-1 antibodies, anti-PD-L1,        anti-CTLA-4, anti-LAG-3, anti-4-1BB, anti-GITR, anti-CD27,        anti-BLTA, anti-OX40, anti-VEGF, anti-TNF-α, anti-IL-2,        anti-GpIIb/IIIa, anti-CD-52, anti-CD20, anti-RSV,        anti-HER2/neu(erbB2), anti-TNF receptor, anti-EGFR antibodies,        monoclonal antibodies or antibody fragments, examples include:        cetuximab, panitumumab, infliximab, basiliximab, bevacizumab        (Avastin®), abciximab, daclizumab, gemtuzumab, alemtuzumab,        rituximab (Mabthera®), palivizumab, trastuzumab, etanercept,        adalimumab, nimotuzumab    -   EGFR inihibitors such as erlotinib, cetuximab and gefitinib    -   anti-angiogenic agents such as bevacizumab (Avastin®)    -   anti-cancer vaccines such as Sipuleucel-T (Provenge®)

In one embodiment the chemotherapeutic agent is an anti-PD-1 antibody oran anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-4-1BB, anti-GITR,anti-CD27, anti-BLTA, anti-OX40, anti-VEGF, anti-TNF-α, anti-IL-2,anti-GpIIb/IIIa, anti-CD-52, anti-CD20, anti-RSV, anti-HER2/neu(erB2)anti-TNF receptor, anti-EGFR antibody. In some embodiments, thechemotherapeutic agent is an immune checkpoint inhibitor orcostimulation molecule.

Further chemotherapeutic drugs may be selected from: 13-cis-RetinoicAcid, 2-Chlorodeoxyadenosine, 5-Azacitidine 5-Fluorouracil,6-Mercaptopurine, 6-Thioguanine, Abraxane®, Accutane®, Actinomycin-DAdriamycin®, Adrucil®, Afinitor®, Agrylin®, AlaCort®, Aldesleukin,Alemtuzumab, Alimta®, Alitretinoin, Alkaban-AQ®, Alkeran®,All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin,Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole,Arabinosylcytosine, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®,Arsenic Trioxide, Asparaginase, ATRA Avastin®, Azacitidine, BCG, BCNU,Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU®,Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, CalciumLeucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, Carac™,Carboplatin, Carmustine, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU®,Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor,Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cytadren®,Cytarabine Cytosar-U®, Cytoxan®, Dacogen®, Dactinomycin, DarbepoetinAlfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride,Daunorubicin Liposomal, DaunoXome®, Decadron®, Decitabine,Delta-Cortef®, Deltasone®, Denileukin, Diftitox, DepoCyt™,Dexamethasone, Dexamethasone Acetate, Dexamethasone Sodium Phosphate,Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil®,Doxorubicin, Doxorubicin Liposomal, Droxia™, DTIC, DTIC-Dome®,Duralone®, Eligard™, Ellence™, Eloxatin™, Elspar®, Emcyt®, Epirubicin,Epoetin Alfa, Erbitux®, Erlotinib, Erwinia L-asparaginase, Estramustine,Ethyol Etopophos®, Etoposide, Etoposide Phosphate, Eulexin®, Everolimus,Evista®, Exemestane, Faslodex®, Femara®, Filgrastim, Floxuridine,Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluoxymesterone,Flutamide, Folinic Acid, FUDR®, Fulvestrant, Gefitinib, Gemcitabine,Gemtuzumab ozogamicin, Gleevec™, Gliadel® Wafer, Goserelin,Granulocyte—Colony Stimulating Factor, Granulocyte Macrophage ColonyStimulating Factor, Herceptin®, Hexadrol, Hexalen®, Hexamethylmelamine,HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®, Hydrocortisone,Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate,Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan,Idamycin®, Idarubicin, Ifex®, IFN-alpha, Ifosfamide, IL-11, IL-2,Imatinib mesylate, Imidazole Carboxamide, Interferon alfa, InterferonAlfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A®(interferon alfa-2b), Iressa®, Irinotecan, Isotretinoin, Ixabepilone,Ixempra™, Kidrolase®, Lanacort®, Lapatinib, L-asparaginase, LCR,Lenalidomide, Letrozole, Leucovorin, Leukeran™, Leukine™, Leuprolide,Leurocristine, Leustatin™, Liposomal Ara-C, Liquid Pred®, Lomustine,L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®, Matulane®, Maxidex™,Mechlorethamine, Mechlorethamine Hydrochloride, Medralone®, Medrol®,Megace®, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna,Mesnex™, Methotrexate, Methotrexate Sodium, Methylprednisolone,Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC,MTX, Mustargen®, Mustine, Mutamycin®, Myleran®, Mylocel™, Mylotarg®,Navelbine®, Nelarabine, Neosar®, Neulasta™, Neumega®, Neupogen®,Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®,Novantrone®, Octreotide, Octreotide acetate, Oncospar®, Oncovin®,Ontak®, Onxal™, Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel,Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin®,Paraplatin®, Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim,PEG-INTRON™, PEG-L-asparaginase, PEMETREXED, Pentostatin, PhenylalanineMustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®,Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with CarmustineImplant Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®,Rituximab, Roferon-A® (Interferon Alfa-2a), Rubex®, Rubidomycinhydrochloride, Sandostatin® Sandostatin LAR®, Sargramostim,Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCEL™, STI-571, Streptozocin,SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®,Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA,Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®,Thiophosphoamide, Thioplex®, Thiotepa™, TICE®, Toposar®, Topotecan,Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin,Trexall™, Trisenox®, TSPA, TYKERB®, VCR, Vectibix™, Velban®, Velcade®,VePesid®, Vesanoid®, Viadur™, Vidaza®, Vinblastine, Vinblastine Sulfate,Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB,VM-26™, Vorinostat, VP-16™, Vumon®, Xeloda®, Zanosar®, Zevalin™,Zinecard®, Zoladex®, Zoledronic acid, Zolinza®, Zometa®.

Routes of Administration

Antibodies, antigen binding fragments, polypeptides and othertherapeutic agents, medicaments and pharmaceutical compositionsaccording to aspects of the present invention may be formulated foradministration by a number of routes, including but not limited to,parenteral, intravenous, intra-arterial, intramuscular, subcutaneous,intradermal intratumoral and oral. Antibodies, antigen bindingfragments, polypeptides and other therapeutic agents, may be formulatedin fluid or solid form. Fluid formulations may be formulated foradministration by injection to a selected region of the human or animalbody.

Dosage Regime

Multiple doses of the antibody, antigen binding fragment or polypeptidemay be provided. One or more, or each, of the doses may be accompaniedby simultaneous or sequential administration of another therapeuticagent.

Multiple doses may be separated by a predetermined time interval, whichmay be selected to be one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or31 days, or 1, 2, 3, 4, 5, or 6 months. By way of example, doses may begiven once every 7, 14, 21 or 28 days (plus or minus 3, 2, or 1 days).

Kits

In some aspects of the present invention a kit of parts is provided. Insome embodiments the kit may have at least one container having apredetermined quantity of the antibody, antigen binding fragment orpolypeptide. The kit may provide the antibody, antigen binding fragmentor polypeptide in the form of a medicament or pharmaceuticalcomposition, and may be provided together with instructions foradministration to a patient in order to treat a specified disease orcondition. The antibody, antigen binding fragment or polypeptide may beformulated so as to be suitable for injection or infusion to a tumor orto the blood.

In some embodiments the kit may further comprise at least one containerhaving a predetermined quantity of another therapeutic agent (e.g.anti-infective agent or chemotherapy agent). In such embodiments, thekit may also comprise a second medicament or pharmaceutical compositionsuch that the two medicaments or pharmaceutical compositions may beadministered simultaneously or separately such that they provide acombined treatment for the specific disease or condition. Thetherapeutic agent may also be formulated so as to be suitable forinjection or infusion to a tumor or to the blood.

Subjects

The subject to be treated may be any animal or human. The subject ispreferably mammalian, more preferably human. The subject may be anon-human mammal, but is more preferably human. The subject may be maleor female. The subject may be a patient. A subject may have beendiagnosed with a disease or condition requiring treatment, or besuspected of having such a disease or condition.

Protein Expression

Molecular biology techniques suitable for the producing polypeptidesaccording to the invention in cells are well known in the art, such asthose set out in Sambrook et al., Molecular Cloning: A LaboratoryManual, New York: Cold Spring Harbor Press, 1989

The polypeptide may be expressed from a nucleotide sequence. Thenucleotide sequence may be contained in a vector present in a cell, ormay be incorporated into the genome of the cell.

A “vector” as used herein is an oligonucleotide molecule (DNA or RNA)used as a vehicle to transfer exogenous genetic material into a cell.The vector may be an expression vector for expression of the geneticmaterial in the cell. Such vectors may include a promoter sequenceoperably linked to the nucleotide sequence encoding the gene sequence tobe expressed. A vector may also include a termination codon andexpression enhancers. Any suitable vectors, promoters, enhancers andtermination codons known in the art may be used to express polypeptidesfrom a vector according to the invention. Suitable vectors includeplasmids, binary vectors, viral vectors and artificial chromosomes (e.g.yeast artificial chromosomes).

In this specification the term “operably linked” may include thesituation where a selected nucleotide sequence and regulatory nucleotidesequence (e.g. promoter and/or enhancer) are covalently linked in such away as to place the expression of the nucleotide sequence under theinfluence or control of the regulatory sequence (thereby forming anexpression cassette). Thus a regulatory sequence is operably linked tothe selected nucleotide sequence if the regulatory sequence is capableof effecting transcription of the nucleotide sequence. Whereappropriate, the resulting transcript may then be translated into adesired protein or polypeptide.

Any cell suitable for the expression of polypeptides may be used forproducing peptides according to the invention. The cell may be aprokaryote or eukaryote. Suitable prokaryotic cells include E. coli.Examples of eukaryotic cells include a yeast cell, a plant cell, insectcell or a mammalian cell. In some cases the cell is not a prokaryoticcell because some prokaryotic cells do not allow for the samepost-translational modifications as eukaryotes. In addition, very highexpression levels are possible in eukaryotes and proteins can be easierto purify from eukaryotes using appropriate tags. Specific plasmids mayalso be utilised which enhance secretion of the protein into the media.

Methods of producing a polypeptide of interest may involve culture orfermentation of a cell modified to express the polypeptide. The cultureor fermentation may be performed in a bioreactor provided with anappropriate supply of nutrients, air/oxygen and/or growth factors.Secreted proteins can be collected by partitioning culturemedia/fermentation broth from the cells, extracting the protein content,and separating individual proteins to isolate secreted polypeptide.Culture, fermentation and separation techniques are well known to thoseof skill in the art.

Bioreactors include one or more vessels in which cells may be cultured.Culture in the bioreactor may occur continuously, with a continuous flowof reactants into, and a continuous flow of cultured cells from, thereactor. Alternatively, the culture may occur in batches. The bioreactormonitors and controls environmental conditions such as pH, oxygen, flowrates into and out of, and agitation within the vessel such that optimumconditions are provided for the cells being cultured.

Following culture of cells that express the polypeptide of interest,that polypeptide is preferably isolated. Any suitable method forseparating polypeptides/proteins from cell culture known in the art maybe used. In order to isolate a polypeptide/protein of interest from aculture, it may be necessary to first separate the cultured cells frommedia containing the polypeptide/protein of interest. If thepolypeptide/protein of interest is secreted from the cells, the cellsmay be separated from the culture media that contains the secretedpolypeptide/protein by centrifugation. If the polypeptide/protein ofinterest collects within the cell, it will be necessary to disrupt thecells prior to centrifugation, for example using sonification, rapidfreeze-thaw or osmotic lysis. Centrifugation will produce a pelletcontaining the cultured cells, or cell debris of the cultured cells, anda supernatant containing culture medium and the polypeptide/protein ofinterest.

It may then be desirable to isolate the polypeptide/protein of interestfrom the supernatant or culture medium, which may contain other proteinand non-protein components. A common approach to separatingpolypeptide/protein components from a supernatant or culture medium isby precipitation. Polypeptides/proteins of different solubility areprecipitated at different concentrations of precipitating agent such asammonium sulfate. For example, at low concentrations of precipitatingagent, water soluble proteins are extracted. Thus, by adding increasingconcentrations of precipitating agent, proteins of different solubilitymay be distinguished. Dialysis may be subsequently used to removeammonium sulfate from the separated proteins.

Other methods for distinguishing different polypeptides/proteins areknown in the art, for example ion exchange chromatography and sizechromatography. These may be used as an alternative to precipitation, ormay be performed subsequently to precipitation.

Once the polypeptide/protein of interest has been isolated from cultureit may be necessary to concentrate the protein. A number of methods forconcentrating a protein of interest are known in the art, such asultrafiltration or lyophilisation.

Sequence Identity

Alignment for purposes of determining percent amino acid or nucleotidesequence identity can be achieved in various ways known to a person ofskill in the art, for instance, using publicly available computersoftware such as ClustalW 1.82. T-coffee or Megalign (DNASTAR) software.When using such software, the default parameters, e.g. for gap penaltyand extension penalty, are preferably used. The default parameters ofClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap ExtensionPenalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=-1, Protein/DNAGAPDIST=4.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

Throughout this specification, including the claims which follow, unlessthe context requires otherwise, the word “comprise,” and variations suchas “comprises” and “comprising,” will be understood to imply theinclusion of a stated integer or step or group of integers or steps butnot the exclusion of any other integer or step or group of integers orsteps.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Ranges may be expressedherein as from “about” one particular value, and/or to “about” anotherparticular value. When such a range is expressed, another embodimentincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by theuse of the antecedent “about,” it will be understood that the particularvalue forms another embodiment.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1. Light chain variable domain sequences for anti-TIM-3 antibody169, 1 H9, 1H10, 2C7, 2F4, 2G6, 1D9, 1F4, 2C8 (human IgG4). CDRs areunderlined and shown separately.

FIG. 2. Heavy chain variable domain sequences for anti- TIM-3 antibodyclones 169, 1 H9, 1H10, 2C7, 2F4, 2G6, 1D9, 1F4, 2C8 (human IgG4). CDRsare underlined and shown separately.

FIG. 3. Table showing light chain and heavy chain CDR sequences foranti-TIM-3 antibody clones 1B9, 1H9, 1H10, 2C7, 2F4, 2G6, 1D9, 1F4, 2C8.

FIG. 4. Nucleotide and encoded amino acid sequences of heavy and lightchain variable domain sequences for anti-TIM-3 antibody clones 1B9, 1H9,1H10, 2C7, 2F4, 2G6, 1D9, 1F4, 2C8 (human IgG4).

FIG. 5. Table showing off rates of clones 1B9, 1H9, 1H10, 2C7, 2F4, 2G6,1D9, 1F4, 2C8.

FIG. 6. Chart showing effect of anti-TIM-3 clone 2C7-anti CD3 bispecificantibody on purified T cells and acute myeloid leukemia (AML) cells. AMLcells were mixed with purified T cells at a 1:1 ratio and the antibodywas added at various concentrations. After 24-hour incubation, lysis wasmeasured.

FIG. 7. Chart showing CD34 specific killing effect of anti-TIM-3 clone2C7-anti CD3 bispecific antibody on CD34+ cells in AML biopsies (i.e.AML stem cells). After selection, CD34+ cells (samples>99% CD34+ purity)were mixed with purified T cells at a 1:1 ratio and the antibody wasadded at various concentrations. After 24-hour incubation, lysis wasmeasured.

FIG. 8. Schematic drawing of the tandem single chain bispecific antibodyformat.

Examples

Isolation of Anti-Human TIM-3 Antibodies

Anti-TIM-3 antibodies were isolated from a human antibody phage displaylibrary via in vitro selection in a 3-round bio-panning process.

Basically, streptavidin-magnetic beads were coated with biotinylatedhuman TIM-3 and used to fish-out anti-TIM-3-specific phages usingmagnetic sorting. Some steps to get rid of potential anti-biotinantibodies were added in the selection process.

After a small-scale induction in HB2151 cells, Fab antibodies werescreened by ELISA. Briefly, ELISA plates were coated with human Tim-3and blocked with a solution of casein. After extensive washes in PBSTween-20, supernatants from induction plates were transferred into theELISA plates in the presence of 7% milk in PBS. After 90 minutes at roomtemperature under agitation and extensive washes, a goat anti-human Fabantibody coupled to HRP was added. One hour later, plates were washedand TMB substrate added. The reaction was stopped with 1M HCl andoptical density measured at 450nm with a reference at 670nm. Antibodiesgiving an absorbance >0.1 were selected as positive. A first clonalityscreening was performed by DNA fingerprinting; clonality was thenconfirmed by sequencing.

Affinity Maturation

One clone showing a good profile in neutralising TIM-3 in vitro wasfurther engineered. After reverting its sequence to a germline-likeframework, this antibody went through affinity maturation by CDRengineering. Selection of specific affinity-matured anti-TIM-3antibodies was conducted by phage display, and screening by ELISA.

In order to select some leads amongst the ELISA-positive clones, TIM-3association/dissociation profiles were analysed by Surface PlasmonResonance (SPR). Briefly, human TIM-3 coupled to Fc was immobilised onsensor chip and the antibodies were then applied as a flow onto thechip. Association and dissociation rates were recorded using a ProteOnXPR36 bioanalyser (Biorad). Nine clones (1B9, 1H9, 1H10, 2C7, 2F4, 2G6,1D9, 1F4, 2C8) were selected based on their rapid/strong association toTIM-3 and their slow dissociation from the protein (FIG. 5).

Specific Killing by Bispecific Anti-TIM-3, Anti-CD3 Antibody of AcuteMyeloid Leukemia Cells from Patients

Anti-TIM-3 clone 2C7 was used to construct a bi-specific antibody ableto engage T cells on one side (specificity for CD3) and target TIM-3 onthe other side. The bispecific antibody comprises two single chainvariable Fragments (scFvs) as a fusion protein. One of the scFvscomprises the VH and VL sequences for clone 2C7 (i.e. SEQ ID NOs: 4 and24), and the other scFv comprises the V_(H) and V_(L) sequences for ananti-CD3 antibody clone.

The format for the tandem single chain bispecific antibody is shown inFIG. 8.

Anti-TIM-3 clone 2C7, in tandem single-chain bispecific format withanti-CD3(anti-TIM-3 clone 2C7-anti CD3 bispecific antibody), was thentested to assess its ability to kill Acute Myeloid Leukamia (AML) cellsobtained from AML patients' biopsies.

Briefly, purified T cells were mixed with AML cells obtained frompatients refractory to 3 lines of chemotherapy treatment, at a ratio of1:1. The bispecific antibody was added at various concentrations, andthe mixture was incubated for 24 hours. After incubation, lysis of AMLcells was measured.

The results are shown in FIG. 6. Anti-TIM-3 clone 2C7-anti CD3bispecific antibody proved to be potent in killing AML cells fromchemotherapy-refractory patients ex vivo.

The clone 2C7-anti CD3 bispecific antibody was then tested on AML stemcells, i.e. cells within the AML biopsies that express high levels ofCD34. After selection, CD34+ cells (samples>99% CD34+ purity) were mixedwith purified T cells at a 1:1 ratio and the antibody was added atvarious concentrations. After 24-hour incubation, lysis was measured.

The results are shown in FIG. 7. Anti-TIM-3 clone 2C7-anti CD3bispecific antibody showed an ability to kill AML stem cells ex vivo, atmedium to high concentrations.

1-57. (canceled)
 58. A method of treating cancer in a patient sufferingfrom a cancer, comprising administering an effective amount of anantibody that binds to TIM-3, or antigen binding fragment thereof, tothe patient, wherein the antibody or antigen binding fragment comprises:(i) at least one light chain variable region incorporating the followingCDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2: (SEQ ID NO: 11)GNNWRPS LC-CDR3: (SEQ ID NO: 12) EAWDYYVAAGY;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 32)GYVAGSDA;

OR (ii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 13) DSWDSADASGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 33)GYVAGFDD;

OR (iii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 14) DSWDYDYAAGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61)  GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 34)GYVAGFDS;

OR (iv) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 15) DSWDSYLAAGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 35)GYVAGYDY;

OR (v) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 16) ESWDYDYASGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 36)GYVAGSDA;

OR (vi) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 17) DSWDSSDSSGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 37)GYVAGFDS;

OR (vii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 18) EAWDSAYAAGS;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 38)GYYAGDDY;

OR (viii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 19) DSWDAALSAGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 39)GYVAGYDY;

OR (ix) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 20) ESWDAAAAAGY;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 40)GYVAGSDA.


59. The method according to claim 58, wherein the heavy chain variableregion has at least 85% sequence identity to the heavy chain variableregion sequence of one of SEQ ID NOs: 21 to 29 (FIG. 2), and the lightchain variable region has at least 85% sequence identity to the lightchain variable region sequence of one of: SEQ ID NO:1 to 9 (FIG. 1). 60.The method according to claim 58, wherein: (i) the heavy chain variableregion has at least 85% sequence identity to the heavy chain variableregion sequence of SEQ ID NO:21, and the light chain variable region hasat least 85% sequence identity to the light chain variable regionsequence of SEQ ID NO:1; OR (ii) the heavy chain variable region has atleast 85% sequence identity to the heavy chain variable region sequenceof SEQ ID NO:22, and the light chain variable region has at least 85%sequence identity to the light chain variable region sequence of SEQ IDNO:2; OR (iii) the heavy chain variable region has at least 85% sequenceidentity to the heavy chain variable region sequence of SEQ ID NO:23,and the light chain variable region has at least 85% sequence identityto the light chain variable region sequence of SEQ ID NO:3; OR (iv) theheavy chain variable region has at least 85% sequence identity to theheavy chain variable region sequence of SEQ ID NO:24, and the lightchain variable region has at least 85% sequence identity to the lightchain variable region sequence of SEQ ID NO:4; OR (v) the heavy chainvariable region has at least 85% sequence identity to the heavy chainvariable region sequence of SEQ ID NO:25, and the light chain variableregion has at least 85% sequence identity to the light chain variableregion sequence of SEQ ID NO:5; OR (vi) the heavy chain variable regionhas at least 85% sequence identity to the heavy chain variable regionsequence of SEQ ID NO:26, and the light chain variable region has atleast 85% sequence identity to the light chain variable region sequenceof SEQ ID NO:6; OR (vii) the heavy chain variable region has at least85% sequence identity to the heavy chain variable region sequence of SEQID NO:27, and the light chain variable region has at least 85% sequenceidentity to the light chain variable region sequence of SEQ ID NO:7; OR(viii) the heavy chain variable region has at least 85% sequenceidentity to the heavy chain variable region sequence of SEQ ID NO:28,and the light chain variable region has at least 85% sequence identityto the light chain variable region sequence of SEQ ID NO:8; OR (ix) theheavy chain variable region has at least 85% sequence identity to theheavy chain variable region sequence of SEQ ID NO:29, and the lightchain variable region has at least 85% sequence identity to the lightchain variable region sequence of SEQ ID NO:9.
 61. The method accordingto claim 58, wherein the antibody or the antigen binding fragment is abispecific antibody or a bispecific antigen binding fragment of theantibody, further comprising an antigen binding domain which binds to atarget protein other than TIM-3.
 62. The method according to claim 61,wherein the antigen binding domain which binds to a target protein otherthan TIM-3 binds to CD3 or a CD3 polypeptide.
 63. The method accordingto claim 58, wherein the antigen binding fragment is a Fab fragment orscFv fragment.
 64. The method according to claim 58, wherein theantibody comprises a human constant region selected from IgG1, IgG2,IgG3 and IgG4.
 65. The method according to claim 58, wherein the canceris a cancer of a tissue selected from the group consisting of: lung,kidney, bladder, liver, stomach, cervix, naso pharynx, oral cavity,oesophagus, larynx, salivary gland, tongue, tonsil, trachea, skin, blood, colon and breast.
 66. The method according to claim 58, wherein thecancer is selected from the gr oup consisting of lung cancer, non-smallcell lung cancer (NSCLC), renal cancer, renal c ell carcinoma, bladdercancer, bladder carcinoma, liver cancer, hepatoma, stomach cancer,cervical cancer, nasopharyngeal cancer, oral cavity cancer, oesophagealcancer, laryngeal cancer, salivary gland cancer, tongue cancer, tonsilcancer, tracheal cancer, skin cancer, m elanoma, metastatic melanoma,haematologic cancer, lymphoma, Hodgkin's lymphoma, col on cancer, coloncarcinoma and breast cancer.
 67. A method of treating an infectiousdisease in a patient suffering from an infectious disease, comprisingadministering an effective amount of an antibody that binds to TIM-3, orantigen binding fragment thereof, to the patient, wherein the antibodyor antigen binding fragment comprises: (i) at least one light chainvariable region incorporating the following CDRs: LC-CDR1:(SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2: (SEQ ID NO: 11) GNNWRPS LC-CDR3:(SEQ ID NO: 12) EAWDYYVAAGY;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 32)GYVAGSDA;

OR (ii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 13) DSWDSADASGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 33)GYVAGFDD;

OR (iii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO:14) DSWDYDYAAGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 34)GYVAGFDS;

OR (iv) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 15) DSWDSYLAAGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 35)GYVAGYDY;

OR (v) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO:16) ESWDYDYASGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 36)GYVAGSDA;

OR (vi) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 17) DSWDSSDSSGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 37)GYVAGFDS;

OR (vii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 18) EAWDSAYAAGS;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 38)GYYAGDDY;

OR (viii) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 19) DSWDAALSAGV;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 39)GYVAGYDY;

OR (ix) at least one light chain variable region incorporating thefollowing CDRs: LC-CDR1: (SEQ ID NO: 10) SGSSSNIGNNYVS LC-CDR2:(SEQ ID NO: 11) GNNWRPS LC-CDR3: (SEQ ID NO: 20) ESWDAAAAAGY;

and at least one heavy chain variable region incorporating the followingCDRs: HC-CDR1: (SEQ ID NO: 61) GYYWS, or (SEQ ID NO: 30) GGSFSGYYWSHC-CDR2: (SEQ ID NO: 31) EINHSGSTNYNPSLKS HC-CDR3: (SEQ ID NO: 40)GYVAGSDA.


68. The method according to claim 67, wherein the heavy chain variableregion has at least 85% sequence identity to the heavy chain variableregion sequence of one of SEQ ID NOs: 21 to 29 (FIG. 2), and the lightchain variable region has at least 85% sequence identity to the lightchain variable region sequence of one of: SEQ ID NO:1 to 9 (FIG. 1). 69.The method according to claim 67, wherein: (i) the heavy chain variableregion has at least 85% sequence identity to the heavy chain variableregion sequence of SEQ ID NO:21, and the light chain variable region hasat least 85% sequence identity to the light chain variable regionsequence of SEQ ID NO:1; OR (ii) the heavy chain variable region has atleast 85% sequence identity to the heavy chain variable region sequenceof SEQ ID NO:22, and the light chain variable region has at least 85%sequence identity to the light chain variable region sequence of SEQ IDNO:2; OR (iii) the heavy chain variable region has at least 85% sequenceidentity to the heavy chain variable region sequence of SEQ ID NO:23,and the light chain variable region has at least 85% sequence identityto the light chain variable region sequence of SEQ ID NO:3; OR (iv) theheavy chain variable region has at least 85% sequence identity to theheavy chain variable region sequence of SEQ ID NO:24, and the lightchain variable region has at least 85% sequence identity to the lightchain variable region sequence of SEQ ID NO:4; OR (v) the heavy chainvariable region has at least 85% sequence identity to the heavy chainvariable region sequence of SEQ ID NO:25, and the light chain variableregion has at least 85% sequence identity to the light chain variableregion sequence of SEQ ID NO:5; OR (vi) the heavy chain variable regionhas at least 85% sequence identity to the heavy chain variable regionsequence of SEQ ID NO:26, and the light chain variable region has atleast 85% sequence identity to the light chain variable region sequenceof SEQ ID NO:6; OR (vii) the heavy chain variable region has at least85% sequence identity to the heavy chain variable region sequence of SEQID NO:27, and the light chain variable region has at least 85% sequenceidentity to the light chain variable region sequence of SEQ ID NO:7; OR(viii) the heavy chain variable region has at least 85% sequenceidentity to the heavy chain variable region sequence of SEQ ID NO:28,and the light chain variable region has at least 85% sequence identityto the light chain variable region sequence of SEQ ID NO:8; OR (ix) theheavy chain variable region has at least 85% sequence identity to theheavy chain variable region sequence of SEQ ID NO:29, and the lightchain variable region has at least 85% sequence identity to the lightchain variable region sequence of SEQ ID NO:9.
 70. The method accordingto claim 67, wherein the antibody or the antigen binding fragment is abispecific antibody or a bispecific antigen binding fragment of theantibody, further comprising an antigen binding domain which binds to atarget protein other than TIM-3.
 71. The method according to claim 70,wherein the antigen binding domain which binds to a target protein otherthan TIM-3 binds to CD3 or a CD3 polypeptide.
 72. The method accordingto claim 67, wherein the antigen binding fragment is a Fab fragment orscFv fragment.
 73. The method according to claim 67, wherein theantibody comprises a human constant region selected from IgG1, IgG2,IgG3 and IgG4.